Nanoemulsion therapeutic compositions and methods of using the same

ABSTRACT

The present invention relates to therapeutic nanoemulsion compositions and to methods of utilizing the same. In particular, nanoemulsion compositions are described herein that find use in the treatment and/or prevention of infection (e.g., respiratory infection (e.g., associated with cystic fibrosis)), in burn wound management, and in immunogenic compositions (e.g., comprising a  Burkholderia  antigen) that generate an effective immune response (e.g., against a bacterial species of the genus  Burkholderia ) in a subject administered the immunogenic composition. Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic and preventative medicine), industrial, and research applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/567,571, filed Sep. 25, 2009, now U.S. Pat. No. 8,962,026,issued Feb. 24, 2015, which claims the benefit of Provisional PatentApplication No. 61/100,559, filed Sep. 26, 2008, each of which arehereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

FIELD OF THE INVENTION

The present invention relates to therapeutic nanoemulsion compositionsand to methods of utilizing the same. In particular, nanoemulsioncompositions are described herein that find use in the treatment and/orprevention of infection (e.g., respiratory infection (e.g., associatedwith cystic fibrosis)), in burn wound management, and in immunogeniccompositions (e.g., comprising a Burkholderia antigen) that generate aneffective immune response (e.g., against a bacterial species of thegenus Burkholderia) in a subject administered the immunogeniccomposition. Compositions and methods of the present invention find usein, among other things, clinical (e.g. therapeutic and preventativemedicine), industrial, and research applications.

BACKGROUND OF THE INVENTION

Bacterial infection caused by opportunistic and/or pathogenic bacteria(e.g., Burkholderia cepacia, Staphylococcus aureus and Pseudomonasaeruginosa) is a major problem in both the developed and undevelopedportions of the world. For example, certain types of individuals areprone to respiratory infection (e.g., by bacteria (e.g., opportunisticbacteria), viruses, fungi and/or parasites) including theimmunocompromised, elderly, cancer chemotherapy patients, individualssuffering from asthma, individual suffering from genetically inheriteddisease (e.g., cystic fibrosis) and virally infected individuals (e.g.,infected with influenza virus, respiratory syncytial virus (RSV),adenovirus and/or human immunodeficiency virus). Similarly, wounds(e.g., burn wounds) present on a subject provide an ideal location forbacterial growth and survival.

As the use of conventional pharmaceutical antibiotics has increased formedical, veterinary and agricultural purposes, there has been aconcurrent emergence of antibiotic-resistant strains of pathogenicbacteria.

A need exists to develop alternative strategies of antibacterialtreatment. For example, there exists a need for new compositions andmethods of treating or preventing bacterial infection (e.g., bacteremia)caused by strains of bacteria unsusceptible to current forms ofantibacterial treatments.

SUMMARY OF THE INVENTION

The present invention relates to therapeutic nanoemulsion compositionsand to methods of utilizing the same. In particular, nanoemulsioncompositions are described herein that find use in the treatment and/orprevention of infection (e.g., respiratory infection (e.g., associatedwith cystic fibrosis)), in burn wound management, and in immunogeniccompositions (e.g., comprising a Burkholderia antigen) that generate aneffective immune response (e.g., against a bacterial species of thegenus Burkholderia) in a subject administered the immunogeniccomposition. Compositions and methods of the present invention find usein, among other things, clinical (e.g. therapeutic and preventativemedicine), industrial, and research applications.

Accordingly, in some embodiments, the present invention provides animmunogenic composition comprising a nanoemulsion and one or moreBurkholderia antigens (e.g., Burkholderia cenocepacia antigens). In someembodiments, the nanoemulsion comprises an aqueous phase, an oil phase,and a solvent. The present invention is not limited by the type ofnanoemulsion composition. Indeed, a variety of nanoemulsion compositionsfind use in the present invention including, but not limited to, thosedescribed herein. The present invention is not limited by the one ormore Burkholderia antigens utilized in the immunogenic compositions andmethods of the invention. Indeed, a variety of Burkholderia antigens maybe utilized including, but not limited to, Burkholderia bacteriainactivated and/or killed by nanoemulsion (NE), killed and/orinactivated Burkholderia bacteria (e.g., via mixing with alcohol (e.g.,ethanol)), whole cell lysates of a Burkholderia bacteria, one or moreisolated, purified and/or recombinant Burkholderia proteins and/orprotein fragments, or other type of Burkholderia antigen describedherein. In a preferred embodiment, the Burkholderia antigen comprisesouter membrane lipoprotein A (OmpA). In some embodiments, the OmpAprotein comprises the amino acid sequence of SEQ ID NO: 1. In someembodiments, the OmpA protein comprises an amino acid sequencecomprising an amino acid sequence of SEQ ID NOS:2-16. In someembodiments, the OmpA protein is from Burkholderia cepacia. However, thepresent invention is not so limited. Indeed, a Burkholderia antigen foruse in an immunogenic composition of the invention may be from anynumber of Burkholderia species or related species including, but notlimited to, B. cenocepacia, B. dolosa, B. multivorans, B. ambifaria, B.vietnamiensis, B. ubonensis, B. thailandensis, B. graminis, B.oklahomensis, B. pseudomallei, B. xenovorans, B. phytofirmans, B.phymatum, R. metallidurans, R. eutropha, R. solanacearum. In someembodiments, an immunogenic composition comprising a Burkholderiaantigen comprises one or more adjuvants (e.g., cholera toxin (CT)). Thepresent invention is not limited to any particular adjuvant and any oneor more adjuvants described herein find use in a composition of theinvention including but not limited to adjuvants that skew toward a Th1and/or Th2 type immune responses described herein. In some embodiments,the nanoemulsion is W₈₀5EC, although the present invention is not solimited. For example, in some embodiments, the nanoemulsion is selectedfrom one of the nanoemulsion formulations described herein. In someembodiments, the composition comprises between 1-50% nanoemulsionsolution, although greater and lesser amounts also find use in theinvention. For example, in some embodiments, the immunogenic compositioncomprises about 1.0%-10%, about 10%-20%, about 20%-30%, about 30%-40%,about 40%-50%, about 50%-60% or more nanoemulsion solution. In someembodiments, the immunogenic composition comprises about 10%nanoemulsion solution. In some embodiments, the immunogenic compositioncomprises about 15% nanoemulsion solution. In some embodiments, theimmunogenic composition comprises about 20% nanoemulsion solution. Insome embodiments, the immunogenic composition comprises about 12%nanoemulsion solution. In some embodiments, the immunogenic compositioncomprises about 8% nanoemulsion solution. In some embodiments, theimmunogenic composition comprises about 5% nanoemulsion solution. Insome embodiments, the immunogenic composition comprises about 2%nanoemulsion solution. In some embodiments, the immunogenic compositioncomprises about 1% nanoemulsion solution. In some embodiments, animmunogenic composition (e.g., that is administered to a subject inorder to generate a Burkholderia specific immune response in thesubject) comprises about 0.05-5000 μg of immunogen (e.g., recombinantand/or purified Burkholderia OmpA protein). In some embodiments, eachdose (e.g., of a composition comprising a NE and an immunogen (e.g.,administered to a subject to induce an immune response (e.g., aprotective immune response (e.g., protective immunity))) comprises about0.05-5000 μg of the immunogen (e.g., recombinant and/or purifiedBurkholderia OmpA protein) and comprises about 0.05-5000 μg of anotherimmunogen (e.g., recombinant and/or purified protein, adjuvant (e.g.,cholera toxin), etc.). In some embodiments, each dose will comprise1-500 μg, in some embodiments, each dose will comprise 350-750 μg, insome embodiments, each dose will comprise 50-200 μg, in someembodiments, each dose will comprise 25-75 μg of immunogen (e.g.,recombinant and/or purified protein (e.g., Burkholderia antigen). Insome embodiments, each dose comprises an amount of the immunogensufficient to generate an immune response. An effective amount of theimmunogen in a dose need not be quantified, as long as the amount ofimmunogen generates an immune response in a subject when administered tothe subject. In some embodiments, the composition is stable (e.g., atroom temperature (e.g., for 12 hours, one day, two days, three days,four days, a week, two weeks, three weeks, a month, two months, threemonths, four months, five months, six months, 9 months, a year or more).In some embodiments, the immunogenic composition comprises apharmaceutically acceptable carrier. The present invention is notlimited to any particular pharmaceutically acceptable carrier. Indeed,any suitable carrier may be utilized including but not limited to thosedescribed herein. In some embodiments, the immunogen comprises aBurkholderia product (e.g., including, but not limited to, a protein,peptide, polypeptide, nucleic acid, polysaccharide, or a membranecomponent derived from the Burkholderia). In some embodiments, theimmunogen and the nanoemulsion are combined in a single vessel.

In some embodiments, the present invention provides a method of inducingan immune response to Burkholderia (e.g., Burkholderia cepacia) in asubject comprising: providing a subject and an immunogenic compositioncomprising a nanoemulsion and an immunogen, wherein the immunogencomprises a Burkholderia antigen and administering the composition tothe subject under conditions such that the subject generates aBurkholderia specific immune response. The present invention is notlimited by the route chosen for administration of a composition of thepresent invention. In some preferred embodiments, administering theimmunogenic composition comprises contacting a mucosal surface of thesubject with the composition. In some embodiments, the mucosal surfacecomprises nasal mucosa. In some embodiments, inducing an immune responseinduces immunity to Burkholderia in the subject. In some embodiments,the immunity comprises systemic immunity. In some embodiments, theimmunity comprises mucosal immunity. In some embodiments, the immuneresponse comprises altered (e.g., enhanced) cytokine expression in thesubject. In some embodiments, the immune response comprises an IgGresponse (e.g., a systemic IgG response) to Burkholderia in the subject.In some embodiments, the immunity protects the subject from displayingsigns or symptoms of disease caused by Burkholderia. In someembodiments, the immunity protects the subject from challenge with asubsequent exposure to live Burkholderia. In some embodiments, thecomposition further comprises an adjuvant. In some embodiments, thesubject is a human. In some embodiments, inducing an immune responseinduces immunity to one or more species of Burkholderia in the subject.In some embodiments, inducing an immune response induces immunity to twoor more species of Burkholderia in the subject. In some embodiments,inducing immunity to Burkholderia comprises systemic immunity. In someembodiments, immunity comprises mucosal immunity. In some embodiments,the immune response comprises altered (e.g., increased) cytokineexpression in the subject. In some embodiments, the immune responsecomprises a systemic IgG response to the immunogen. In some embodiments,the immune response comprises a mucosal IgA response to the immunogen.In some embodiments, each dose comprises an amount of Burkholderiaantigen sufficient to generate an immune response to Burkholderia. Aneffective amount of the Burkholderia antigen is a dose that need not bequantified, as long as the amount of Burkholderia antigen generates animmune response in a subject when administered to the subject.

Thus, in some embodiments, the present invention provides an immunogeniccomposition comprising a nanoemulsion and a Burkholderia antigen (e.g.,wherein the Burkholderia antigen comprises outer membrane lipoprotein A(OmpA)). In some embodiments, the OmpA protein comprises the amino acidsequence of SEQ ID NO: 1. In some embodiments, the OmpA proteincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:2-16. In some embodiment, the OmpA protein is fromBurkholderia cepacia. In some embodiments, the present inventionprovides a method of making a vaccine comprising the steps of mixingantigens to make an immunogenic composition comprising a nanoemulsionand a Burkholderia antigen (e.g., wherein the Burkholderia antigencomprises outer membrane lipoprotein A (OmpA)) and adding apharmaceutically acceptable excipient. The present invention alsoprovides a use of the immunogenic composition comprising a nanoemulsionand a Burkholderia antigen (e.g., wherein the Burkholderia antigencomprises outer membrane lipoprotein A (OmpA)) in the manufacture of avaccine for treatment or prevention of Burkholderia infection. In someembodiments, the present invention provides a use of the immunogeniccomposition comprising a nanoemulsion and a Burkholderia antigen (e.g.,wherein the Burkholderia antigen comprises outer membrane lipoprotein A(OmpA)) in the treatment or prevention of Burkholderia infection. Thepresent invention also provides a method of preparing an immune globulinfor use in prevention or treatment of Burkholderia infection comprisingthe steps of immunizing a recipient with an immunogenic compositioncomprising a nanoemulsion and a Burkholderia antigen (e.g., wherein theBurkholderia antigen comprises outer membrane lipoprotein A (OmpA) e.g.,a Burkholderia vaccine) and isolating immune globulin from therecipient.

The present invention also provides compositions and methods fortreating burn wounds. For example, in some embodiments, the presentinvention provides a method of treating a burn wound comprising:providing a subject harboring a burn wound; and a composition comprisinga nanoemulsion; and administering the composition comprising ananoemulsion to the burn wound, wherein the administering reducesinflammation at the site of the burn wound. In some embodiments,administering the composition comprising a nanoemulsion reduces,attenuates and/or prevents bacterial growth in the burn wound. In someembodiments, administering the composition comprising a nanoemulsionreduces tissue edema at the site of the burn wound. In some embodiments,administering the composition comprising a nanoemulsion reducesintravascular hypovolemia at the site of the burn wound. The presentinvention is not limited by the type of nanoemulsion utilized foradministration to a burn wound. For example, in some embodiments, ananoemulsion comprising distilled water; polysorbate (Tween) 20;glycerine; soybean oil; either cetylpyridinium chloride (CPC) orbenzalkonium chloride or alkyl dimethyl benzyl ammonium chloride (BTC824), or a combination thereof; and ethylenediaminetetraacetic acid(EDTA) is administered to a burn wound. In some embodiments,administering the composition comprising a nanoemulsion reduces theoccurrence of shock, pulmonary dysfunction, abdominal or extremitycompartment syndrome, or cardiac failure in the subject. In someembodiments, the composition is co-administered with an antimicrobialagent or an anti-inflammatory agent. The present invention is notlimited by the type of antimicrobial agent or anti-inflammatory agentutilized. Indeed, a variety of antimicrobial agents or ananti-inflammatory agents may be co-administered with the compositioncomprising a nanoemulsion including but not limited to those describedherein. In some embodiments, the antimicrobial agent is an antibiotic.In some embodiments, the anti-inflammatory agent is silver nitrate(AgNO₃), silver sulfadiazine, mafenide acetate, nanocrystallineimpregnated silver dressings, a p38 MAPK inhibitor or otheranti-inflammatory agent.

In some embodiments, the present invention provide a compositioncomprising a nanoemulsion, wherein the nanoemulsion comprises: about 19%by volume distilled water; about 5% by volume polysorbate (Tween) 20;about 8% by volume glycerine; about 64% by volume soybean oil; about 4%by volume of either cetylpyridinium chloride (CPC) or benzalkoniumchloride or alkyl dimethyl benzyl ammonium chloride, or a combinationthereof; and about 0.07% by volume ethylenediaminetetraacetic acid(EDTA). In some embodiments, the composition comprises a nanoemulsioncomprising droplets the have an average diameter selected from the groupcomprising less than about 1000 nm, less than about 950 nm, less thanabout 900 nm, less than about 850 nm, less than about 800 nm, less thanabout 750 nm, less than about 700 nm, less than about 650 nm, less thanabout 600 nm, less than about 550 nm, less than about 500 nm, less thanabout 450 nm, less than about 400 nm, less than about 350 nm, less thanabout 300 nm, less than about 250 nm, less than about 200 nm, less thanabout 150 nm, less than about 100 nm, greater than about 50 nm, greaterthan about 70 nm, greater than about 125 nm, and any combinationthereof.

In some embodiments, the composition further comprises an antimicrobialagent or anti-inflammatory agent. The present invention is not limitedby the type of antimicrobial agent or anti-inflammatory agent utilized.Indeed, a variety of antimicrobial agents or an anti-inflammatory agentsmay be co-administered with the composition comprising a nanoemulsionincluding but not limited to those described herein. In someembodiments, the antimicrobial agent is an antibiotic. In someembodiments, the anti-inflammatory agent is silver nitrate (AgNO₃),silver sulfadiazine, mafenide acetate, nanocrystalline impregnatedsilver dressings, a p38 MAPK inhibitor or other anti-inflammatory agent.In some embodiments, the present invention provides a method of treatingan infection present on and/or within a burn wound comprisingadministering the composition to the infection under conditions suchthat the composition kills, attenuates growth of and/or eliminatesbacteria associated with the infection. The present invention is notlimited by the type of bacteria associated with infection of a burnwound treated with a nanoemulsion of the invention. In some embodiments,bacteria associated with infection comprise Staphylococcus aureus. Insome embodiments, the Staphylococcus aureus are antibiotic resistant. Insome embodiments, the bacteria associated with the infection comprisePseudomonas aeruginosa. The present invention is not limited by the typeof burn wound treated. In some embodiments, the burn wound is asuperficial burn wound, a partial thickness burn wound, or other type ofburn wound.

The present invention is not limited by the type of nanoemulsionutilized. Indeed, a variety of nanoemulsions are contemplated to beuseful in the present invention. For example, in some preferredembodiments, the nanoemulsion (e.g., for pulmonary administration (e.g.,to treat or prevent respiratory infection) or for burn wound treatment)comprises an oil-in-water emulsion, the oil-in-water emulsion comprisinga discontinuous oil phase distributed in an aqueous phase, a firstcomponent comprising a solvent (e.g., an alcohol or glycerol), and asecond component comprising a surfactant or a halogen-containingcompound. The aqueous phase can comprise any type of aqueous phaseincluding, but not limited to, water (e.g., diH₂O, distilled water, tapwater) and solutions (e.g., phosphate buffered saline solution). The oilphase can comprise any type of oil including, but not limited to, plantoils (e.g., soybean oil, avocado oil, flaxseed oil, coconut oil,cottonseed oil, squalene oil, olive oil, canola oil, corn oil, rapeseedoil, safflower oil, and sunflower oil), animal oils (e.g., fish oil),flavor oil, water insoluble vitamins, mineral oil, and motor oil. Insome preferred embodiments, the oil phase comprises 30-90 vol % of theoil-in-water emulsion (i.e., constitutes 30-90% of the total volume ofthe final emulsion), more preferably 50-80%. While the present inventionin not limited by the nature of the alcohol component, in some preferredembodiments, the alcohol is ethanol or methanol. Furthermore, while thepresent invention is not limited by the nature of the surfactant, insome preferred embodiments, the surfactant is a polysorbate surfactant(e.g., TWEEN 20, TWEEN 40, TWEEN 60, and TWEEN 80), apheoxypolyethoxyethanol (e.g., TRITON X-100, X-301, X-165, X-102, andX-200, and TYLOXAPOL) or sodium dodecyl sulfate. Likewise, while thepresent invention is not limited by the nature of the halogen-containingcompound, in some preferred embodiments, the halogen-containing compoundcomprises a cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides,tetradecyltrimethylammonium halides, cetylpyridinium chloride,cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride,cetylpyridinium bromide, cetyltrimethylammonium bromide,cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide,dodecyltrimethylammonium bromide, or tetrad ecyltrimethylammoniumbromide. Nanoemulsions of the present invention may further comprisethird, fourth, fifth, etc. components. In some preferred embodiments, anadditional component is a surfactant (e.g., a second surfactant), agermination enhancer, a phosphate based solvent (e.g., tributylphosphate), a neutramingen, L-alanine, ammonium chloride, trypticase soybroth, yeast extract, L-ascorbic acid, lecithin, p-hyroxybenzoic acidmethyl ester, sodium thiosulate, sodium citrate, inosine, sodiumhyroxide, dextrose, and polyethylene glycol (e.g., PEG 200, PEG 2000,etc.). In some embodiments, the oil-in-water emulsion comprises aquaternary ammonium compound. In some preferred embodiments, theoil-in-water emulsion has no detectable toxicity to plants or animals(e.g., to humans). In other preferred embodiments, the oil-in-wateremulsion causes no detectable irritation to plants or animals (e.g., tohumans). In some embodiments, the oil-in-water emulsion furthercomprises any of the components described above. Quaternary ammoniumcompounds include, but are not limited to, N-alkyldimethyl benzylammonium saccharinate, 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol;1-Decanaminium, N-decyl-N, N-dimethyl-, chloride (or) Didecyl dimethylammonium chloride; 2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ehyl dimethylbenzyl ammonium chloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyldimethyl benzyl ammonium chloride; alkyl 1 or 3benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride; alkylbis(2-hydroxyethyl) benzyl ammonium chloride; alkyl demethyl benzylammonium chloride; alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride(100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (50%C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (100% C14); alkyldimethyl benzyl ammonium chloride (100% C16); alkyl dimethyl benzylammonium chloride (41% C14, 28% C12); alkyl dimethyl benzyl ammoniumchloride (47% C12, 18% C14); alkyl dimethyl benzyl ammonium chloride(55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride (58% C14,28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12);alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14); alkyldimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyl dimethylbenzyl ammonium chloride (65% C12, 25% C14); alkyl dimethyl benzylammonium chloride (67% C12, 24% C14); alkyl dimethyl benzyl ammoniumchloride (67% C12, 25% C14); alkyl dimethyl benzyl ammonium chloride(90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93% C14, 4%C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18); alkyldimethyl benzyl ammonium chloride (and) didecyl dimethyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids);alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzylammonium chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethylammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride; alkyldimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyldimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as inthe fatty acids of soybean oil); alkyl dimethyl ethylbenzyl ammoniumchloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyldimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3%C18); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1%C12); alkyl trimethyl ammonium chloride (90% C18, 10% C16);alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); Di-(C8-10)-alkyldimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyldimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkylmethyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride;diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammoniumchloride; dodecyl bis (2-hydroxyethyl) octyl hydrogen ammonium chloride;dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyldinethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazoliniumchloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;myristalkonium chloride (and) Quat RNIUM 14;N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethylbenzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammoniumchloride; n-tetradecyl dimethyl benzyl ammonium chloride monohydrate;octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammoniumchloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride;oxydiethylenebis (alkyl dimethyl ammonium chloride); quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride; trimethoxysily propyldimethyl octadecyl ammonium chloride; trimethoxysilyl quats, trimethyldodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethylbenzyl ammoniumchloride; n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyldimethyl benzyl ammonium chloride; n-tetradecyl dimethyl ethyylbenzylammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride. Insome embodiments, the emulsion lacks any antimicrobial substances (i.e.,the only antimicrobial composition is the emulsion itself). In someembodiments, the nanoemulsion is X8P. In some embodiments, thenanoemulsion is P₄₀₇5EC. In some embodiments, administration of ananoemulsion of the present invention protects the subject fromdisplaying signs or symptoms of disease or infection (e.g., caused byone or microbes and/or pathogens (e.g., pathogenic bacteria)) residingin the pulmonary system (e.g., Burkhoderia cepacia and Pseudomonasaeruginosa)). In some embodiments, administration of a nanoemulsion(e.g., pulmonary administration (e.g., to treat or prevent pulmonaryinfection)) protects a subject from morbidity and/or mortalityassociated with respiratory infection. In some embodiments, the subjectis a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows killing of Burkholderia cepacia in PBS by 20% Nanemulsion(NE) alone or with 20 mM EDTA in 10 minutes.

FIG. 2 shows killing of B. cepacia in PBS by 20% NE alone or with 20 mMEDTA in 20 minutes.

FIG. 3 shows killing of B. cepacia in PBS by 20% NE alone or with 20 mMEDTA in 40 minutes.

FIG. 4 shows (FIG. 4A) killing of B. cepacia in PBS by 20% NE alone orwith 20 mM EDTA in 40 minutes; (FIG. 4B) killing of B. cepacia in PBS by20% NE alone or with 20 mM EDTA in 60 minutes; and (FIG. 4C) killing ofB. cepacia in PBS by 20% NE alone or with 20 mM EDTA in 60 minutes.

FIG. 5 shows killing of B. cepacia in hypertonic saline (6% NaCl) at 15and 30 Minutes by NE alone and NE with EDTA.

FIG. 6 shows killing of B. cepacia and P. aeruginosa in 7% NaCl within15 min.

FIG. 7 shows killing of B. cepacia and P. aeruginosa (Mixed-culture) in7% NaCl within 15 minutes.

FIG. 8 shows a microtiter serial dilution MIC assay. Numbers above theplate indicate the concentration (μg/ml CPC) of P₄₀₇5EC added to eachcolumn of wells. The bacterial species added to wells in each row isindicated on the left. The MICs for these strains (top to bottom) are≦15.6 μg/ml, 125 μg/ml, and 31.2 μg/ml.

FIG. 9 shows distribution of P₄₀₇5EC MICs for 150 Burkholderia andnon-Burkholderia strains

FIG. 10 shows a time-kill study of P₄₀₇5EC activity andconcentration-dependent inhibition of P₄₀₇5EC activity by CF sputum. (A)10⁵ CFU/ml of B. multivorans ATCC 17616 were exposed to variousconcentrations of P₄₀₇5EC around the MIC (62.5 μg/ml) and viablebacterial counts were determined at the times indicated. (B) 10⁵ CFU/mlof B. multivorans ATCC 17616 were exposed to various concentrations ofP₄₀₇5EC in the presence of three concentrations of CF sputum for 24 hrbefore viable bacterial counts were determined. Numbers and symbols ininset boxes indicate concentrations of P₄₀₇5EC (μg/ml CPC) used.

FIG. 11 shows in vitro activity of P₄₀₇5EC.

FIG. 12 shows in vitro activity of P₄₀₇5EC against biofilm bacteria andin the presence of CF sputum.

FIG. 13 shows influence of P₄₀₇5EC on B. cepacia, P. aeruginosa and E.coli individual biofilms.

FIG. 14 shows influence of P₄₀₇5EC on mixed biofilms.

FIG. 15 shows influence of P₄₀₇5EC on mixed biofilms.

FIG. 16 shows influence of P₄₀₇5EC on escape colonies of B. cepaciabiofilms.

FIG. 17 shows serial, two-fold dilutions of P₄₀₇5EC in one embodiment ofthe invention.

FIG. 18 shows age specific prevalence of respiratory infections in CFpatients.

FIG. 19A shows a Fractional Inhibitory Concentration Index;

FIG. 19B shows a graph demonstrating the relationship betweenantagonism, indifference, and synergy for Antimicrobial A(micrograms/mL) and Antimicrobial B (micrograms/mL);

FIGS. 20A-D show figures of electron microscopy. FIG. 20A shows 0 min,no treatment control; FIG. 20B shows 10 minutes after treatment; FIG.20C shows 20 minutes after treatment; and FIG. 20D shows 30 minutesafter treatment.

FIG. 21 shows topical application of nanoemulsion reduces P. aeruginosagrowth in burn wounds. Male Sprague-Dawley rats received partialthickness burn wounds. At 8 hours post-injury, animals were inoculatedwith 10⁶ CFU P. aeruginosa. At 16 and 24 hours post-burn the animalswere treated with topical saline (Control), placebo (W₂₀5GBA₂ED withoutbenzalkonium chloride), W₂₀5GBA₂ED (nanoemulsion), or Sulfamylon(mafenide acetate). At 32 hours, animals were sacrificed, skin samplesobtained and homogenized, plated, and CFUs counted. The scatter plotrepresents cultured CFUs for each individual animal. The median valuefor each group is plotted as a horizontal line. There was minimalpathogen growth in 12 out of 23 of the NB-201 treated animals. Amajority of the control (29/32) and placebo (9/12) animals with burninjury had evidence of wound infection based on a positive quantitativewound culture with significantly more bacteria present in the wound thanthose animals treated with W₂₀5GBA₂ED. p<0.0001, Kruskal-Wallis test,p<0.05 for saline vs. W₂₀5GBA₂ED, placebo vs. W₂₀5GBA₂ED, and saline vs.Sulfamylon, Dunn's multiple comparison test.

FIG. 22 shows nanoemulsion treatment following burn injury attenuatesdermal proinflammatory cytokine expression. Groups were sham, burn, andburn with W₂₀5GBA₂ED treatment (n=8-10 per group). A) IL-1β (p=0.02,1-way ANOVA), B) IL-6 (p=0.8), C) TNF-α (p=0.5), D) CINC-1 (p=0.04), E)CINC-3 (p=0.005), and F) Myeloperoxidase (p=0.07). Burn injured animalstreated with nanoemulsion had decreased levels of IL-1β (1773±516 vs.5625±1743 μg/mL) and CINC-3 (225±66 vs. 1589±527 pg/mL) when compared tountreated partial thickness burned animals. *p<0.05, t-test or Tukey'smultiple comparison test.

FIG. 23 shows nanoemulsion treatment following burn wound infection withP. aeruginosa attenuates dermal proinflammatory cytokine expression. Allanimals received burn injury and groups were saline, placebo,W₂₀5GBA₂ED, and Sulfamylon (n=10-30 per group). A) IL-1β (p=0.001, 1-wayANOVA), B) IL-6 (p=0.07), C) TNF-α (p=0.3), D) CINC-1 (p=0.8), E) CINC-3(p=0.4), and F) Myeloperoxidase (p=0.0001). Burn wound infected animalstreated with nanoemulsion had decreased levels of IL-1β (1007±157 vs.3054±499 pg/mL) and IL-6 (244±51 vs. 485±73 pg/mL). The nanoemulsion andSulfamylon treatment groups demonstrated reduced dermal neutrophilsequestration when compared to saline and placebo as evidenced onmyeloperoxidase assay (W₂₀5GBA₂ED: 0.09±0.02, Sulfamylon: 0.08±0.02,Saline: 0.40±0.06, Placebo: 0.45±0.11 μg/mL). *p<0.05, Tukey's multiplecomparison test.

FIG. 24 shows burn injury upregulates dermal TGF-β expression andtreatment with nanoemulsion in the setting of burn wound infectiondecreases the level of TGF-β in the wound. Dermal levels of theanti-inflammatory cytokines IL-10 and TGF-β were measured in the burnwound 32 hours after thermal injury in animals treated withnanoemulsion, and in animals exposed to bacteria and treated withnanoemulsion or Sulfamylon. A) IL-10 (p=0.4, 1-way ANOVA), and B) TGF-β(p=0.0001). Partial thickness burn increased the presence of dermalTGF-β as compared to sham (624±55 vs. 232±17 pg/mL). Treatment of aninfected burn wound with W₂₀5GBA₂ED significantly reduced the dermallevel of TGF-β compared to the untreated burn group (404±43 vs. 624±55pg/mL). Sulfamylon treatment did not suppress the level of IL-10 orTGF-β in the infected burn wound. *p<0.05, Tukey's multiple comparisontest.

FIG. 25 shows cross sectional histology of burn skin following infectionwith P. aeruginosa and treatment with saline or nanoemulsion. Both viewsof skin are 32 hours after burn injury. A) Representative section fromsaline (control) treated animal (Hematoxylin and eosin×40). B)Representative cross-section from W₂₀5GBA₂ED (nanoemulsion) treatedanimal (Hematoxylin and eosin×40).

FIG. 26 shows evans blue assay to quantify capillary leak and tissueedema. Evans blue is a dye that binds to serum albumin and can bequantitated to determine vascular permeability. Topical nanoemulsiontreatment resulted in less capillary leak following thermal injury andbacterial inoculation of the wound when compared to saline treatedcontrol animals (1.26±0.05 vs. 1.93±0.24 μg Evans blue/mg tissue, n=8per group). *p=0.02, t-test.

FIG. 27 shows photomicrographs of partial thickness burned skin withfluorescence labeled TUNEL staining to detect hair follicle cellapoptosis. Skin samples were harvested at 12 hours post-burn. Treatmentwas performed at 0 and 8 hours following thermal injury. All images areat 40× magnification.

FIG. 28 shows TUNEL assay for hair follicle cell apoptosis following apartial thickness burn injury. Skin samples from sham, burn+saline(control), burn+placebo (W₂₀5GBA₂ED without benzalkonium chloride), andburn+W₂₀5GBA₂ED were sectioned for fluorescein-labeled TUNEL assay.Treatment was performed at time 0 and 8 hours post injury. No bacterialinoculation of the burned skin was done in this experiment. There was asignificant reduction in the degree of hair follicle apoptosis amongburn injured and treated animals for tissue samples harvested at 12hours post injury (p=0.006, 1-way ANOVA). Differences were found forsham vs. burn+saline, burn+saline vs. burn+placebo, and burn+saline vs.burn+W₂₀5GBA₂ED (*p<0.05, Tukey's multiple comparison test).

FIG. 29 shows characterization of the OMP preparation. A) Agarose gelelectrophoresis and ethidium bromide-staining Lane 1. DNA ladder; Lane2. Whole Cell lysate (WCL) before separation by high speedcentrifugation; Lane 3. Supernatant following the 100,000×g spin (lane Bin B, C, & D); Lane 4. Crude OMP preparation (lane C in B, C, & D); Lane5. Endotoxin (ET) depleted OMP fraction (lane E in B, C, & D).Volumetrically-loaded silver stain B) and western blot of OMPpreparation (C-D). Lane A. Protein from the supernatant produced afterthe 6000×g centrifugation was loaded; Lane B. An equal volume of thesupernatant from the 100,000×g centrifugation; Lane C. Crude OMP (there-suspended pellet fraction of the 100,000×g spin); Lanes D-J.Endotoxin-depleted OMP fractions (the successive flow-through portionsof the endotoxin column); Lanes K-N. Endotoxin column retentantfractions (the successive fluid regenerated from the column after theaddition of sodium deoxycholate). C) Western blot probed with serum frommice immunized with the endotoxin-depleted OMP-NE preparation. D)Western blot probed with serum from mice immunized with the OMP in PBSpreparation.

FIG. 30 shows antibody responses against B. cenocepacia OMP. A) ELISAresults of the IgG response in serum post-immunization with the OMPpreparation with or without nanoemulsion. Serum anti-OMP IgG antibodyconcentrations are presented as mean of endpoint titers in individualsera±SEM. * indicates a statistical difference (p<0.05) in the anti-OMPIgG titers. B) Mucosal antibodies sIgA and IgG against the OMP afternasal vaccine with or without nanoemulsion. sIgA and IgG were measuredin BAL solution. The OD levels were normalized to total protein contentwithin the samples.

FIG. 31 shows type of cellular immune responses induced by nasal OMP-NEvaccine. A) Serum from mice immunized with 5 μg OMP mixed with either NE(OMP-NE) or with PBS (OMP-PBS) was analyzed for antibody subtypedistribution. The results are presented as ratio of the specificsubclass IgG to the overall IgG titer. *: indicates statisticaldifference (p<0.05) between IgG2b and IgG1 subtypes. B) Cytokineprofiling of splenocytes of mice immunized with 5 μg. mixed with eitherNE (OMP-NE) or with PBS (OMP-PBS) Data is represented as fold change±SEMcomparing OMP-activated versus non activated splenocytes and isnormalized to responses in non-vaccinated mice. *: indicates statisticaldifference (p<0.05) between OMP-NE and OMP in PBS groups.

FIG. 32 shows identification of protein epitopes and LPS in OMPpreparations. Lane 1. Protein ladder; Lane 2. Crude OMP preparation;Lane 3. Proteinase K digested crude OMP preparation; Lane 4.Endotoxin-depleted OMP; Lane 5; Proteinase K digested endotoxin-depletedOMP. Western blots were probed with serum from either OMP in PBS orOMP-NE immunized mice or with a monoclonal anti-Pseudomonas mallei LPSantibody as indicated.

FIG. 33 shows serum neutralization assay. Percent reduction of B.cenocepacia or B. multivorans cfu plotted against samples with naïveserum. A statistical difference in B. cenocepacia neutralizing activitywas observed between serum from mice immunized with OMP-NE and serumfrom mice immunized with OMP in PBS formulations (p=9.9×10⁻⁵ for 5 ugOMP-NE and 0.03 for 15 ug OMP-NE). B. multivorans cross-neutralizingactivity was observed between serum from mice immunized with OMP-NE andserum from naïve mice (p=0.04). * indicates a statistically significant(p<0.05) difference in neutralizing activity between OMP-NE and OMP inPBS. ** indicates a statistically significant (p<0.05) difference inneutralizing activity between OMP-NE vaccinated and naïve mice.

FIG. 34 shows pulmonary and splenic colonization assay. A) Pulmonarytissue associated and B) Splenic tissue associated cfu determined at sixdays following intratracheal challenge of 5×10⁷ cfu of B. cenocepacia.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “microorganism” refers to any species or typeof microorganism, including but not limited to, bacteria, viruses,archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.The term microorganism encompasses both those organisms that are in andof themselves pathogenic to another organism (e.g., animals, includinghumans, and plants) and those organisms that produce agents that arepathogenic to another organism, while the organism itself is notdirectly pathogenic or infective to the other organism.

As used herein, the term “pathogen” refers a biological agent thatcauses a disease state (e.g., infection, sepsis, etc.) in a host.“Pathogens” include, but are not limited to, viruses, bacteria, archaea,fungi, protozoans, mycoplasma, prions, and parasitic organisms.

The terms “bacteria” and “bacterium” refer to all prokaryotic organisms,including those within all of the phyla in the Kingdom Procaryotae. Itis intended that the term encompass all microorganisms considered to bebacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, andRickettsia. All forms of bacteria are included within this definitionincluding cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.Also included within this term are prokaryotic organisms that areGram-negative or Gram-positive. “Gram-negative” and “Gram-positive”refer to staining patterns with the Gram-staining process, which is wellknown in the art. (See e.g., Finegold and Martin, DiagnosticMicrobiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 (1982)).“Gram-positive bacteria” are bacteria that retain the primary dye usedin the Gram stain, causing the stained cells to generally appear darkblue to purple under the microscope. “Gram-negative bacteria” do notretain the primary dye used in the Gram stain, but are stained by thecounterstain. Thus, Gram-negative bacteria generally appear red. In someembodiments, bacteria are continuously cultured. In some embodiments,bacteria are uncultured and existing in their natural environment (e.g.,at the site of a wound or infection) or obtained from patient tissues(e.g., via a biopsy). Bacteria may exhibit pathological growth orproliferation. Examples of bacteria include, but are not limited to,bacterial cells of a genus of bacteria selected from the groupcomprising Salmonella, Shigella, Escherichia, Enterobacter, Serratia,Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella,Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus,Micrococcus, Staphylococcus, Vibrio, Aeromonas, Plessiomonas,Haemophilus, Actinobacillus, Pasteurella, Mycoplasma, Ureaplasma,Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus,Enterococcus, Aerococcus, Gemella, Lactococcus, Leuconostoc, Pedicoccus,Bacillus, Corynebacterium, Arcanobacterium, Actinomyces, Rhodococcus,Listeria, Erysipelothrix, Gardnerella, Neisseria, Campylobacter,Arcobacter, Wolinella, Helicobacter, Achromobacter, Acinetobacter,Agrobacterium, Alcaligenes, Chryseomonas, Comamonas, Eikenella,Flavimonas, Flavobacterium, Moraxella, Oligella, Pseudomonas,Shewanella, Weeksella, Xanthomonas, Bordetella, Franciesella, Brucella,Legionella, Afipia, Bartonella, Calymmatobacterium, Cardiobacterium,Streptobacillus, Spirillum, Peptostreptococcus, Peptococcus, Sarcinia,Coprococcus, Ruminococcus, Propionibacterium, Mobiluncus,Bifidobacterium, Eubacterium, Lactobacillus, Rothia, Clostridium,Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Bilophila,Leptotrichia, Wolinella, Acidaminococcus, Megasphaera, Veilonella,Norcardia, Actinomadura, Norcardiopsis, Streptomyces, Micropolysporas,Thermoactinomycetes, Mycobacterium, Treponema, Borrelia, Leptospira, andChlamydiae.

As used herein, the terms “microorganism” and “microbe” refer to anyspecies or type of microorganism, including but not limited to,bacteria, archaea, fungi, protozoans, mycoplasma, and parasiticorganisms.

As used herein, the term “fungi” is used in reference to eukaryoticorganisms such as molds and yeasts, including dimorphic fungi.

As used herein the terms “disease” and “pathologic condition” are usedinterchangeably, unless indicated otherwise herein, to describe adeviation from the condition regarded as normal or average for membersof a species or group (e.g., humans), and which is detrimental to anaffected individual under conditions that are not inimical to themajority of individuals of that species or group. Such a deviation canmanifest as a state, signs, and/or symptoms (e.g., diarrhea, nausea,fever, pain, blisters, boils, rash, immune suppression, inflammation,etc.) that are associated with any impairment of the normal state of asubject or of any of its organs or tissues that interrupts or modifiesthe performance of normal functions. A disease or pathological conditionmay be caused by or result from contact with a microorganism (e.g., apathogen or other infective agent (e.g., a virus or bacteria)), may beresponsive to environmental factors (e.g., malnutrition, industrialhazards, and/or climate), may be responsive to an inherent defect of theorganism (e.g., genetic anomalies) or to combinations of these and otherfactors.

“Respiratory” and “respiration” refer to the process by which oxygen istaken into the body and carbon dioxide is discharged, through the bodilysystem including the nose, throat, larynx, trachea, bronchi and lungs.

“Respiratory infection” and “pulmonary infection” refer to an infection(e.g., bacterial, viral, fungal, etc.) of the respiratory tract. Inhumans, the respiratory tract comprises the upper respiratory tract(e.g., nose, throat or pharynx, and larynx); the airways (e.g.,: voicebox or larynx, windpipe or trachea, and bronchi); and the lungs (e.g.,bronchi, bronchioles, alveolar ducts, alveolar sacs, and alveoli).

“Respiratory disease”, “pulmonary disease,” “respiratory disorder”,“pulmonary disorder,” “respiratory condition”, “pulmonary condition,”“pulmonary syndrome,” and “respiratory syndrome” refer to any one ofseveral ailments that involve inflammation and affect a component of therespiratory system including especially the trachea, bronchi and lungs.Examples of such ailments include acute alveolar disease, obstructiverespiratory disease (e.g., asthma; bronchitis; and chronic obstructivepulmonary disease, referred to as COPD), upper airway disease (e.g.,such as otitis media, and rhinitis/sinusitis), insterstitial lungdisease, allergy, and respiratory infection (e.g., pneumonia,pneyumocystis carinii, and respiratory syncitial virus (RSV)).

Specific examples of acute alveolar disease include acute lung injury(ALI), acute respiratory distress syndrome (ARDS), meconium aspirationsyndrome (MAS) and respiratory distress syndrome (RDS). ALI isassociated with conditions that either directly or indirectly injure theair sacs of the lung, the alveoli. ALI is a syndrome of inflammation andincreased permeability of the lungs with an associated breakdown of thelungs' surfactant layer. The most serious manifestation of ALI is ARDS.Among the causes of ALI are complications typically associated withcertain major surgeries, mechanical ventilator induced lung injury(often referred to as VILI), smoke inhalation, pneumonia, and sepsis.

The term “subject” as used herein refers to organisms to be treated bythe compositions of the present invention. Such organisms includeanimals (domesticated animal species, wild animals), and humans.

As used herein, the terms “inactivating,” “inactivation” and grammaticalequivalents, when used in reference to a microorganism refer to thekilling, elimination, neutralization and/or reducing the capacity of themicroorganism to infect and/or cause a pathological response and/ordisease in a host.

As used herein, the term “fusigenic” is intended to refer to an emulsionthat is capable of fusing with the membrane of a microbial agent (e.g.,a bacterium or bacterial spore). Specific examples of fusigenicemulsions are described herein.

As used herein, the term “lysogenic” refers to an emulsion (e.g., ananoemulsion) that is capable of disrupting the membrane of a microbialagent (e.g., a virus (e.g., viral envelope) or a bacterium, bacterialspore, or bacterial biofilm). In preferred embodiments of the presentinvention, the presence of a lysogenic and a fusigenic agent in the samecomposition produces an enhanced inactivating effect compared to eitheragent alone. Methods and compositions using this improved antimicrobialcomposition are described in detail herein.

The term “nanoemulsion,” as used herein, includes dispersions ordroplets, as well as other lipid structures that can form as a result ofhydrophobic forces that drive apolar residues (i.e., long hydrocarbonchains) away from water and drive polar head groups toward water, when awater immiscible oily phase is mixed with an aqueous phase. These otherlipid structures include, but are not limited to, unilamellar,paucilamellar, and multilamellar lipid vesicles, micelles, and lamellarphases.

As used herein, the terms “contact,” “contacted,” “expose,” and“exposed,” when used in reference to a nanoemulsion and a livemicroorganism, refer to bringing one or more nanoemulsions into contactwith a microorganism (e.g., a pathogen) such that the nanoemulsion killand/or attenuate growth of the microorganism or pathogenic agent, ifpresent. The present invention is not limited by the amount or type ofnanoemulsion used for microorganism killing and/or growth attenuation. Avariety of nanoemulsion that find use in the present invention aredescribed herein and elsewhere (e.g., nanoemulsions described in U.S.Pat. Apps. 20020045667 and 20040043041, and U.S. Pat. Nos. 6,015,832,6,506,803, 6,635,676, and 6,559,189, each of which is incorporatedherein by reference in its entirety for all purposes). Ratios andamounts of nanoemulsion are contemplated in the present inventionincluding, but not limited to, those described herein (e.g., in Examples1-4, the Figures associated therewith and FIG. 17).

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

The term “surfactant” refers to any molecule having both a polar headgroup, which energetically prefers solvation by water, and a hydrophobictail which is not well solvated by water. The term “cationic surfactant”refers to a surfactant with a cationic head group. The term “anionicsurfactant” refers to a surfactant with an anionic head group.

The terms “Hydrophile-Lipophile Balance Index Number” and “HLB IndexNumber” refer to an index for correlating the chemical structure ofsurfactant molecules with their surface activity. The HLB Index Numbermay be calculated by a variety of empirical formulas as described byMeyers, (Meyers, Surfactant Science and Technology, VCH Publishers Inc.,New York, pp. 231-245 [1992]), incorporated herein by reference. As usedherein, the HLB Index Number of a surfactant is the HLB Index Numberassigned to that surfactant in McCutcheon's Volume 1: Emulsifiers andDetergents North American Edition, 1996 (incorporated herein byreference). The HLB Index Number ranges from 0 to about 70 or more forcommercial surfactants. Hydrophilic surfactants with high solubility inwater and solubilizing properties are at the high end of the scale,while surfactants with low solubility in water which are goodsolubilizers of water in oils are at the low end of the scale.

As used herein the term “interaction enhancers” refers to compounds thatact to enhance the interaction of an emulsion with a microorganism(e.g., with a cell wall of a bacteria (e.g., a Gram negative bacteria)or with a viral envelope. Contemplated interaction enhancers include,but are not limited to, chelating agents (e.g.,ethylenediaminetetraacetic acid (EDTA),ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and the like)and certain biological agents (e.g., bovine serum abulmin (BSA) and thelike).

The terms “buffer” or “buffering agents” refer to materials which whenadded to a solution, cause the solution to resist changes in pH.

The terms “reducing agent” and “electron donor” refer to a material thatdonates electrons to a second material to reduce the oxidation state ofone or more of the second material's atoms.

The term “monovalent salt” refers to any salt in which the metal (e.g.,Na, K, or Li) has a net 1+ charge in solution (i.e., one more protonthan electron).

The term “divalent salt” refers to any salt in which a metal (e.g., Mg,Ca, or Sr) has a net 2+ charge in solution.

The terms “chelator” or “chelating agent” refer to any materials havingmore than one atom with a lone pair of electrons that are available tobond to a metal ion.

The term “solution” refers to an aqueous or non-aqueous mixture.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., a composition comprising a nanoemulsion) sufficientto effect a beneficial or desired result (e.g., to treat and/or preventinfection (e.g., through bacterial cell killing and/or prevention ofbacterial cell growth). An effective amount can be administered in oneor more administrations, applications or dosages and is not intended tobe limited to a particular formulation or administration route.

As used herein, the term “a composition for inducing an immune response”refers to a composition that, when administered to a subject (e.g.,once, twice, three times or more (e.g., separated by weeks, months oryears)), stimulates, generates and/or elicits an immune response in thesubject (e.g., resulting in total or partial immunity to a microorganism(e.g., pathogen) capable of causing disease). In preferred embodimentsof the invention, the composition comprises a nanoemulsion and animmunogen. In further preferred embodiments, the composition comprisinga nanoemulsion and an immunogen comprises one or more other compounds oragents including, but not limited to, therapeutic agents,physiologically tolerable liquids, gels, carriers, diluents, adjuvants,excipients, salicylates, steroids, immunosuppressants, immunostimulants,antibodies, cytokines, antibiotics, binders, fillers, preservatives,stabilizing agents, emulsifiers, and/or buffers. An immune response maybe an innate (e.g., a non-specific) immune response or a learned (e.g.,acquired) immune response (e.g. that decreases the infectivity,morbidity, or onset of mortality in a subject (e.g., caused by exposureto a pathogenic microorganism) or that prevents infectivity, morbidity,or onset of mortality in a subject (e.g., caused by exposure to apathogenic microorganism)). Thus, in some preferred embodiments, acomposition comprising a nanoemulsion and an immunogen is administeredto a subject as a vaccine (e.g., to prevent or attenuate a disease(e.g., by providing to the subject total or partial immunity against thedisease or the total or partial attenuation (e.g., suppression) of asign, symptom or condition of the disease.

As used herein, the term “adjuvant” refers to any substance that canstimulate an immune response (e.g., a mucosal immune response). Someadjuvants cause activation of a cell of the immune system (e.g., anadjuvant can cause an immune cell to produce and secrete a cytokine)Examples of adjuvants that can cause activation of a cell of the immunesystem include, but are not limited to, the nanoemulsion formulationsdescribed herein, saponins purified from the bark of the Q. saponariatree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLCfractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.);poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); cholera toxin (CT), and Leishmaniaelongation factor (a purified Leishmania protein; Corixa Corporation,Seattle, Wash.). Traditional adjuvants are well known in the art andinclude, for example, aluminum phosphate or hydroxide salts (“alum”). Insome embodiments, immunogenic compositions described herein areadministered with one or more adjuvants (e.g., to skew the immuneresponse towards a Th1 and/or Th2 type response).

As used herein, the term “an amount effective to induce an immuneresponse” (e.g., of a composition for inducing an immune response),refers to the dosage level required (e.g., when administered to asubject) to stimulate, generate and/or elicit an immune response in thesubject. An effective amount can be administered in one or moreadministrations (e.g., via the same or different route), applications ordosages and is not intended to be limited to a particular formulation oradministration route.

As used herein, the term “under conditions such that said subjectgenerates an immune response” refers to any qualitative or quantitativeinduction, generation, and/or stimulation of an immune response (e.g.,innate or acquired).

As used herein, the term “immune response” refers to any detectableresponse by the immune system of a subject. For example, immuneresponses include, but are not limited to, an alteration (e.g.,increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g.,Th1 or Th2 type cytokines) or chemokine) expression and/or secretion,macrophage activation, dendritic cell activation, T cell (e.g., CD4+ orCD8+ T cell) activation, NK cell activation, and/or B cell activation(e.g., antibody generation and/or secretion). Additional examples ofimmune responses include binding of an immunogen (e.g., antigen (e.g.,immunogenic polypeptide)) to an MHC molecule and induction of acytotoxic T lymphocyte (“CTL”) response, induction of a B cell response(e.g., antibody production), and/or T-helper lymphocyte response, and/ora delayed type hypersensitivity (DTH) response (e.g., against theantigen from which an immunogenic polypeptide is derived), expansion(e.g., growth of a population of cells) of cells of the immune system(e.g., T cells, B cells (e.g., of any stage of development (e.g., plasmacells), and increased processing and presentation of antigen by antigenpresenting cells. An immune response may be to immunogens that thesubject's immune system recognizes as foreign (e.g., non-self antigensfrom microorganisms (e.g., pathogens), or self-antigens recognized asforeign). Thus, it is to be understood that, as used herein, “immuneresponse” refers to any type of immune response, including, but notlimited to, innate immune responses (e.g., activation of Toll receptorsignaling cascade) cell-mediated immune responses (e.g., responsesmediated by T cells (e.g., antigen-specific T cells) and non-specificcells of the immune system) and humoral immune responses (e.g.,responses mediated by B cells (e.g., via generation and secretion ofantibodies into the plasma, lymph, and/or tissue fluids). The term“immune response” is meant to encompass all aspects of the capability ofa subject's immune system to respond to an antigen and/or immunogen(e.g., both the initial response to an immunogen (e.g., a pathogen) aswell as acquired (e.g., memory) responses that are a result of anadaptive immune response).

As used herein, the term “immunity” refers to protection from disease(e.g., preventing or attenuating (e.g., suppression of) a sign, symptomor condition of the disease) upon exposure to a microorganism (e.g.,pathogen) capable of causing the disease. Immunity can be innate (e.g.,non-adaptive (e.g., non-acquired) immune responses that exist in theabsence of a previous exposure to an antigen) and/or acquired (e.g.,immune responses that are mediated by B and T cells following a previousexposure to antigen (e.g., that exhibit increased specificity andreactivity to the antigen)).

As used herein, the terms “immunogen” and “antigen” are usedinterchangeably to refer to an agent (e.g., a microorganism (e.g.,bacterium, virus or fungus) and/or portion or component thereof (e.g., aprotein antigen)) that is capable of eliciting an immune response in asubject. In preferred embodiments, immunogens elicit immunity againstthe immunogen (e.g., microorganism (e.g., pathogen or a pathogenproduct)) when administered in combination with a nanoemulsion of thepresent invention. As used herein, the term Burkholderia antigen refersto a component or product of a bacteria of the genus Burkholderia thatelicits an immune response when administered to a subject. An antigenmay be a component or product derived from an organism (e.g., bacteriaof the genus Burkholderia) including, but not limited to, polypeptides,peptides, proteins, nucleic acids, membrane fractions, andpolysaccharides.

As used herein, the term “enhanced immunity” refers to an increase inthe level of adaptive and/or acquired immunity in a subject to a givenimmunogen (e.g., microorganism (e.g., pathogen)) followingadministration of a composition relative to the level of adaptive and/oracquired immunity in a subject that has not been administered thecomposition.

As used herein, the terms “purified” or “to purify” refer to the removalof contaminants or undesired compounds from a sample or composition. Asused herein, the term “substantially purified” refers to the removal offrom about 70 to 90%, up to 100%, of the contaminants or undesiredcompounds from a sample or composition.

As used herein, the terms “administration” and “administering” refer tothe act of giving a drug, prodrug, or other agent, or therapeutictreatment (e.g., a composition of the present invention) to aphysiological system (e.g., a subject or in vivo, in vitro, or ex vivocells, tissues, and organs).

As used herein, the terms “co-administration” and “co-administering”refer to the administration of at least two agent(s) (e.g., ananoemulsion and one or more other pharmaceutically acceptablesubstances (e.g., a second nanoemulsion)) or therapies to a subject. Insome embodiments, the co-administration of two or more agents ortherapies is concurrent. In other embodiments, a first agent/therapy isadministered prior to a second agent/therapy. In some embodiments,co-administration can be via the same or different route ofadministration. Those of skill in the art understand that theformulations and/or routes of administration of the various agents ortherapies used may vary. The appropriate dosage for co-administrationcan be readily determined by one skilled in the art. In someembodiments, when agents or therapies are co-administered, therespective agents or therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents or therapies lowers the requisite dosage of a potentially harmful(e.g., toxic) agent(s), and/or when co-administration of two or moreagents results in sensitization of a subject to beneficial effects ofone of the agents via co-administration of the other agent. In otherembodiments, co-administration is preferable to treat and/or preventinfection by more than one type of infectious agent (e.g., bacteriaand/or viruses).

As used herein, the term “topically” refers to application of acompositions of the present invention (e.g., a composition comprising ananoemulsion) to the surface of the skin and/or mucosal cells andtissues (e.g., alveolar, buccal, lingual, masticatory, vaginal or nasalmucosa, and other tissues and cells which line hollow organs or bodycavities). Compositions described herein can be applied using anypharmaceutically acceptable method, such as for example, intranasal,buccal, sublingual, oral, rectal, ocular, parenteral (intravenously,intradermally, intramuscularly, subcutaneously, intracisternally,intraperitoneally), pulmonary, intravaginal, locally administered,topically administered, mucosally administered, via an aerosol, or via abuccal or nasal spray formulation. Further, the nanoemulsion vaccinesdescribed herein can be formulated into any pharmaceutically acceptabledosage form, such as a liquid dispersion, gel, aerosol, pulmonaryaerosol, nasal aerosol, ointment, cream, semi-solid dosage form, and asuspension. Further, the composition may be a controlled releaseformulation, sustained release formulation, immediate releaseformulation, or any combination thereof.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse allergic or immunological reactions whenadministered to a host (e.g., an animal or a human). Such formulationsinclude dips, sprays, seed dressings, stem injections, sprays, andmists. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, wetting agents (e.g.,sodium lauryl sulfate), isotonic and absorption delaying agents,disintegrants (e.g., potato starch or sodium starch glycolate), and thelike. Examples of carriers, stabilizers and adjuvants have beendescribed and are known in the art (See e.g., Martin, Remington'sPharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975),incorporated herein by reference).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acomposition of the present invention that is physiologically toleratedin the target subject. “Salts” of the compositions of the presentinvention may be derived from inorganic or organic acids and bases.Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compositions of the inventionand their pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,persulfate, phenylpropionate, picrate, pivalate, propionate, succinate,tartrate, thiocyanate, tosylate, undecanoate, and the like. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

For therapeutic use, salts of the compositions of the present inventionare contemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable composition.

As used herein, the terms “at risk for disease” and “at risk forinfection” refer to a subject that is predisposed to experiencing aparticular disease and/or infection. This predisposition may be genetic(e.g., a particular genetic tendency to experience the disease, such asheritable disorders), or due to other factors (e.g., environmentalconditions, exposures to detrimental compounds present in theenvironment, etc.). Thus, it is not intended that the present inventionbe limited to any particular risk (e.g., a subject may be “at risk fordisease” simply by being exposed to and interacting with other peoplethat carry a risk of transmitting a pathogen), nor is it intended thatthe present invention be limited to any particular disease and/orinfection.

“Nasal application”, as used herein, means applied through the nose intothe nasal or sinus passages or both. The application may, for example,be done by drops, sprays, mists, coatings or mixtures thereof applied tothe nasal and sinus passages.

“Pulmonary application” and “pulmonary administration” refers to anymeans of applying a composition of the present invention to thepulmonary system of a subject. The present invention is not limited toany particular means of administration. Indeed, a variety of means arecontemplated to be useful for pulmonary administration including thosedescribed herein.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of the nanoemulsion compositions ofthe present invention, such delivery systems include systems that allowfor the storage, transport, or delivery of the compositions and/orsupporting materials (e.g., written instructions for using thematerials, etc.) from one location to another. For example, kits includeone or more enclosures (e.g., boxes) containing the relevantnanoemulsions and/or supporting materials. As used herein, the term“fragmented kit” refers to delivery systems comprising two or moreseparate containers that each contain a subportion of the total kitcomponents. The containers may be delivered to the intended recipienttogether or separately. For example, a first container may contain acomposition comprising a nanoemulsion for a particular use, while asecond container contains a second agent (e.g., an antibiotic or sprayapplicator). Indeed, any delivery system comprising two or more separatecontainers that each contains a subportion of the total kit componentsare included in the term “fragmented kit.” In contrast, a “combined kit”refers to a delivery system containing all of the components of acomposition needed for a particular use in a single container (e.g., ina single box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions useful fortreating pulmonary infection. In particular, the present inventionprovides nanoemulsion compositions and methods of using the same totreat bacteria associated with biofilms (e.g., found in pulmonaryinfections). Compositions and methods of the present invention find usein, among other things, clinical (e.g. therapeutic and preventativemedicine), industrial, and research applications.

Several pathogenic microorganisms initiate infection by attaching tomucosal epithelial cells lining the gastro-intestinal, oropharyngeal,respiratory or genito-urinacy tracts. Some pathogens, such as influenzavirus, Bordetella pertussis, or Vibrio cholerae, remain at or within themucosal tissue, while others, such as Salmonella typhi or hepatitis Avirus, possess mechanisms permitting penetration into deeper tissues andspread systemically. Specific and non-specific defense mechanisms of themucous membranes provide first line protection against both types ofpathogen. Non-specific effectors include resident macrophages,antimicrobial peptides, lactoferrin and lysozyme, extremes of pH, bileacids, digestive enzymes, mucus, shedding of epithelial cells, flushingmechanisms (peristalsis, ciliary beating, micturation, etc.) andcompetition from local flora. However, successful pathogens havegenerally evolved means to survive the non-specific defenses present atthe site they infect and it is the secretory immune system which plays amajor role in protecting against diseases caused by a number ofbacterial and viral pathogens, and is probably a major effector againstpathogens that are restricted to mucosal surfaces. For organisms thatspread systemically, both local and systemic immune responses aredesirable for optimum immunity.

As described herein, certain microbes (e.g., bacteria) are able tothrive when a normal and/or healthy immune system does not functionproperly for one reason or another. Cystic fibrosis (CF), asthma, HIVinfection, chemotherapeutic therapy and a host of other conditions leadto malfunctioning and/or attenuation of immune responses that wouldnormally function to protect against and clear microbes capable ofcausing pathology in a healthy subject.

For example, Pseudomonas aeruginosa is an opportunistic pathogen thatinfects the immunocompromised, elderly, cancer chemotherapy patients,and individual suffering from CF. In CF lung disease, P. aeruginosa istrapped in thickened, dehydrated, hypoxic mucus lining in airwayepithelia. Morphologic data suggests that the airway lumen of CFpatients harbor P. aeruginosa biofilms that are characterized asspherical microcolonies.

Other types of microbes can cause pathology in an otherwise healthy hostsubject. For example, colonization of the respiratory tract by theGram-negative coccobacillus Bordetella pertussis results in whoopingcough, also called pertussis, a significant cause of morbidity andmortality of human infants. Two other closely-related isolates ofBordetella have also been found in humans: B. parapertussis and B.bronchiseptica. Molecular genetic analyses suggest that these threeisolates are too closely related to be classified as separate species.(See Gilchrist. M. J. R., 1991, “Bordetella”, in Manual of ClinicalMicrobiology, 5th ed., Balows, A. et al., eds., American Society forMicrobiology, Washington, D.C.). While B. pertussis differs from B.bronchiseptica and B. parapertussis in the nature of the toxins itproduces, B. bronchiseptica and B. parapertussis do produce activetoxins (See Hausman, S. Z. et al., 1996, Infect. Immun. 64: 4020-4026),and there is some evidence to indicate that B. pertussis organisms cancovert to the B. parapertussis phenotype (Gilchrist, M. J. R., 1991,“Bordetella”, in Manual of Clinical Microbiology, 5th ed., Balows, A. etal., eds., American Society for Microbiology, Washington, D.C.).

Thus, the present invention provides compositions and methods for thetreatment of respiratory infections. Compositions and methods of thepresent invention may be used to treat and/or prevent respiratoryinfection (e.g., in cystic fibrosis patients) caused by one or more ofpseudomonas (e.g., P. aeruginosa, P. paucimobilis, P. putida, P.fluorescens, and P. acidovorans), staphylococci, Methicillin-resistantStaphylococcus aureus (MRSA), streptococci (including Streptococcuspneumoniae), Escherichia coli, Klebsiella, Enterobacter, Serratia,Haemophilus, Yersinia pestis, Burkholderia pseudomallei, B. cepacia, B.gladioli, B. multivorans, B. vietnamiensis, Mycobacterium tuberculosis,M. avium complex (MAC)(M. avium and M. intracellulare), M. kansasii, M.xenopi, M. marinum, M. ulcerans, or M. fortuitum complex (M. fortuitumand M. chelonei), Bordetella pertussis, B. parapertussis and B.bronchiseptica. Furthermore, compositions and methods of the presentinvention find use in the treatment and/or prevention of a host ofrespiratory infections (e.g., respiratory infections of the upperrespiratory tract (e.g., nose, ears, sinuses, and throat) and the lowerrespiratory tract (e.g., trachea, bronchial tubes, and lungs)). Severalexamples of microbes that may be treated (e.g., killed and/or attenuatedin growth (e.g., within the respiratory tract of a subject)) areprovided below.

Accordingly, in some embodiments, the present invention provides acomposition comprising a nanoemulsion, and methods of pulmonaryadministration of the same to prevent and/or treat respiratoryinfection. In some embodiments, the nanoemulsion comprisesethylenediaminetetraacetic acid (EDTA). The present invention is notlimited by the amount of EDTA utilized. In some embodiments, 0.01-0.1mM, 0.1-1.0 mM, 1.0-1.5 mM, 1.5-2.5 mM, 2.5-5.0 mM, 5.0-10.0 mM, 10-20mM, 20-30 mM, or 30-50 mM EDTA is used. However, the present inventionis not limited to this amount of EDTA. In some embodiments, less than0.01 mM or more than 50 mM EDTA is utilized. In some embodiments, thecomposition is co-administered with a hypertonic salt (e.g., sodiumchloride) solution (e.g., a 6-7%, 1-3%, 3-6%, 0.1-1%, or more than 7%salt solution (e.g., NaCl solution)). In some embodiments, thecomposition comprises a 20% nanoemulsion solution. In some embodiments,the composition comprises greater than 20% (e.g., 25%, 30%, or more)nanoemulsion solution. In some embodiments, the composition comprisesless than 20% (e.g., 15%, 10% or less) nanoemulsion solution. However,the present invention is not limited to this amount (e.g., percentage)of nanoemusion. For example, in some embodiments, a compositioncomprises less than 10% nanoemulsion. In some embodiments, a compositioncomprises more than 20% nanoemulsion. In some embodiments, thecomposition comprises 10 mM EDTA. In some embodiments, the compositioncomprises 20 mM EDTA. In some embodiments, a composition of the presentinvention comprises any of the nanoemulsions described herein. In someembodiments, a composition comprising a nanoemulsion utilized to treatbacteria (e.g., present in pulmonary space of a subject (e.g., biofilmforming bacteria)) comprises P₄₀₇5EC. In some embodiments, a compositioncomprising a nanoemulsion comprises W₈₀5EC.

Administration of nanoemulsion alone or in combination with EDTA (e.g.,10-20 mM EDTA) was able to achieve complete killing of 10⁶ bacteria inPBS in 60 minutes (See Examples 2-4). In the presence of hypertonicsaline (e.g., 6-7% NaCl), the killing ability of the nanoemulsion wassurprisingly and strikingly enhanced, achieving complete killing within15 minutes while in the presence of 20 mM EDTA (See Example 2). Also,nanoemulsions comprising a lower concentration of EDTA were able toachieve complete killing of bacteria in 30 minutes in the presence ofhypertonic saline.

Thus, in some embodiments, the present invention provides that ananoemulsion composition can be used to kill (e.g., completely) bacteriaover a short time period (e.g., less than 60 minutes, less than 30minutes, or less than 15 minutes).

The present invention also demonstrates that compositions of the presentinvention are able to eradicate a mixed population of bacteria.Moreover, toxicity studies performed during the development ofembodiments of the present invention characterized the nanoemulsioncompositions as being safe and causing no detectable harm to a subject(e.g., no histological changes and/or detectable pathology (See, e.g.,Example 1)).

As described in Examples 3 and 4, compositions comprising nanoemulsionsof the present invention are able to treat (e.g., kill and/or inhibitgrowth of) bacterial species that are generally not virulent in healthypersons, but that are opportunists that cause severe and chronicrespiratory tract infections (e.g., in individuals with cystic fibrosis(CF)). Unremitting infection with these species results in inflammationand progressive lung disease that culminates in pulmonary failure, theleading cause of death for CF patients. Effective therapy of pulmonaryinfection in CF to date has been severely limited by the broad spectrumantimicrobial resistance exhibited by these species, which are among themost drug-resistant bacteria encountered in human infection. The site ofinfection in CF presents another important obstacle to effectivetherapy. Infecting bacteria primarily reside within the airway lumen insputum, airway epithelial surface fluid, and the bronchial mucosa (ref).The penetration of systemically delivered antimicrobials to thisinfected site is generally poor. Treatment is further hampered bybacterial biofilm formation, which is believed to occur in the airwaysof infected patients, and by the exceptionally viscous secretions thatcharacterize the CF respiratory tract.

Although an understanding of a mechanism is not necessary to practicethe present invention, and the present invention is not limited to anyparticular mechanism of action, in some embodiments, a mechanism ofbacterial and/or viral killing utilizing compositions comprisingnanoemulsions of the present invention involves fusion of the emulsionwith microorganism lipid membranes, leading to rapid osmotic disruptionand cell lysis (See, e.g., Hamouda and Baker, J Appl Microbiol. 2000September; 89(3):397-403). Additionally, in some embodiments,electrostatic attraction (e.g., provided by cationic surface charge ofCPC) overcomes the LPS-mediated resistance of gram-negative bacteria toneutral and anionic detergents (See, e.g., Hamouda and Baker, J ApplMicrobiol. 2000 September; 89(3):397-403). In some embodiments,bactericidal activity of a composition comprising a nanoemulsion (e.g.,P₄₀₇5EC (e.g., against gram-negative and/or gram-positive bacteria)) isenhanced by the addition of EDTA. Although an understanding of amechanism is not necessary to practice the present invention, and thepresent invention is not limited to any particular mechanism of action,in some embodiments, EDTA chelates divalent cations that stabilize outermembrane LPS thereby facilitating interactions with the cationicemulsion, an interaction that leads to membrane permeabilization andlysis as well as augmenting transmembrane diffusion of macromolecules(See, e.g., Rabinovich-Guilatt et al., J Drug Target. 2004;12(9-10):623-33, Vaara, Microbiol Rev. 1992 September; 56(3):395-411).

Experiments conducted during development of embodiments of the presentinvention identified P₄₀₇5EC to be stable after nebulization in 7%saline using the PARI LC Plus nebulizer Inhalation of a single dose ofP₄₀₇5EC nebulized in 7% saline was well tolerated by animals. Thus, insome embodiments, the present invention provides compositions comprisingP₄₀₇5EC and hypertonic saline, and methods of using the same (e.g., viainhalation (e.g., post nebulizing the solution)) for treating one ormore types of pulmonary bacterial infections (e.g., to treat bacteriaand/or bacterial biofilms (e.g., that possess resistance to conventionaltreatments) without increasing the already high treatment burden for CFpatients).

For example, experiments conducted during development of embodiments ofthe invention show that compositions comprising nanoemulsions (e.g.,P₄₀₇5EC and saline solution) were effective to treat (e.g., kill and/orinhibit growth of) species within the Burkholderia cepacia complex (Bcc)(See, e.g., Example 3). Infection with Burkholderia cepacia species isparticularly refractory to antimicrobial therapy and associated withincreased rates of morbidity and mortality in CF. Infection with Bcc isalso regarded by many CF care centers as an absolute contraindication tolung transplantation. Compositions comprising nanoemulsions (e.g.,P₄₀₇5EC and saline solution) were effective to treat (e.g., kill and/orinhibit growth of) B. multivorans and B. cenocepacia which account forthe majority of Bcc infection in CF (See, e.g., Reik J Clin Microbiol.2005 June; 43(6):2926-8). Compositions comprising nanoemulsions (e.g.,P₄₀₇5EC and saline solution) were effective to treat (e.g., kill and/orinhibit growth of) B. gladioli, which although not a member of the Bcc,is being recovered with increasing frequency from CF patients. Alsoshown in Example 3, compositions comprising nanoemulsions (e.g.,P₄₀₇5EC) were effective to treat (e.g., kill and/or inhibit growth of)Acinetobacter, which although currently infrequently recovered in CF,appears to be an emerging pathogen in this patient population. The greatmajority (94%) of isolates tested in Example 3 were recovered fromcultures of respiratory specimens from persons with CF. The panel alsoincluded one representative isolate from each of five previouslydescribed so-called epidemic lineages, each of which has been identifiedas infecting multiple CF patients. These included the B. cenocepaciaET12 (Johnson et al., J Clin Microbiol 1994; 32:924-30.), PHDC (Chen etal., J Pediatr 2001; 139:643-9), and Midwest (Coenye and LiPuma, J.Infect. Dis. 185:1454-1462) lineages, as well as the B. multivorans OHBMlineage (Biddick et al., FEMS Microbiol Lett 2003; 228:57-62) and the B.dolosa SLC6 lineage (Biddick et al., FEMS Microbiol Lett 2003;228:57-62). Compositions comprising nanoemulsions (e.g., P₄₀₇5EC andsaline solution) effectively treated (e.g., killed and/or inhibitedgrowth of) each of these different species. Moreover, compositionscomprising nanoemulsions (e.g., P₄₀₇5EC) were effective to treat (e.g.,kill and/or inhibit growth of) strains that were found in previoussusceptibility testing to be multi-drug resistant (e.g., defined asresistant to all antibiotics tested in two of three antibiotic classes:lactams (including carbapenems), aminoglycosides, and quinolones) aswell as panresistant strains (See Example 3). Genotyping analyses of allisolates was performed to confirm that each sample treated was adistinct strain.

As shown in Example 3, P₄₀₇5EC showed very good activity against thestrains tested. With the exception of two B. cenocepacia strains, allstrains in the test panel were inhibited by a P₄₀₇5EC concentration of≦125 μg/ml, or a 1:16 dilution of the starting material; 59% of strainswere inhibited by a concentration of ≦31.2 μg/ml, a 1:64 dilution ofP₄₀₇5EC. Multi-drug resistant or panresistant strains did not showdecreased susceptibility to P₄₀₇5EC, demonstrating MIC₉₀s of 125 μg/mland 62.5 μg/ml, respectively. In general, Burkholderia species tended tobe slightly less susceptible to P₄₀₇5EC than the other species examined.Sixteen of the 18 strains requiring the highest MICs were Burkholderia.Conversely, 33 of the 38 strains with the lowest tested MIC (15.6 μg/ml)were non-Burkholderia species. No striking differences in P₄₀₇5ECactivity was observed among the 10 Burkholderia species examined,although the two strains requiring the highest MICs (250 μg/ml and 500μg/ml) were both B. cenocepacia. Among the non-Burkholderia species,Ralstonia strains were most susceptible (MIC₉₀≦15.6 μg/ml) whileAcinetobacter strains were relatively less susceptible (MIC₉₀=125μg/ml). No evidence was found of tolerance to P₄₀₇5EC among a subset of34 strains for which both MIC and MBC were determined. P₄₀₇5EC killingof planktonically grown bacteria was time- and concentration-dependent;at a concentration 16 times greater than the MIC, complete killing wasachieved within 30 min. Thus, the present invention provides thatrelatively brief exposure of bacteria to P₄₀₇5EC can be utilized toeffect several log decreases in viable bacteria. Thus, a compositioncomprising a nanoemulsion (e.g., P₄₀₇5EC) of the present invention canbe utilized individually, or in combination with other antimicrobials,to kill and/or inhibit growth of bacteria.

Experiments were conducted during development of embodiments of theinvention to assess the activity of P₄₀₇5EC against bacteria grown invitro as biofims. A relatively strict definition of in vitro biofilmformation was employed, as described in Example 3, in an effort toprovide a stringent test of P₄₀₇5EC activity. 12 strains that met thedefinition were tested. As shown in Example 3, the strains representedseveral species and a range of susceptibility to P₄₀₇5EC based onstandard MIC/MBC testing. Although the MBIC and MBEC of P₄₀₇5EC wereincreased compared to the respective MIC and MBC for each strain tested(median four-fold increase in MBIC compared to MIC), all 12 strains wereinhibited or killed by P₄₀₇5EC when grown as biofilms. Only a singlestrain required undiluted P₄₀₇5EC (2000 μg/ml) for eradication of viablebiofilm bacteria. Relative biomass was assessed among the biofilmforming strains spectrophotometrically with crystal violet staining andno correlation was observed between biomass and MBIC/MBEC. In fact,although B. gladioli strain AU10529 produced the greatest biomass amongthe 12 strains tested, the MBIC and MBEC of P₄₀₇5EC for this strain wererelatively low (both 62.5 μg/ml). Conversely, B. cenocepacia strainJ2315 produced relatively little biomass, yet required a P₄₀₇5EC MBEC of1000 μg/ml.

CF sputum, like biofilm, is reported to antagonize the activity ofantibacterial drugs (See, e.g., Hunt et al., Antimicrob AgentsChemother. 1995 January; 39(1):34-9). Glycoproteins such as mucin, whichcompose 2-3% of the dry weight of sputum, and high molecular weight DNAare present in elevated levels resulting in exceptionally viscous sputumthat provides a physical barrier protecting bacteria. In addition, thesemacromolecules bind and sequester antibiotics while small cationicmolecules and the decreased pH of CF sputum block drug penetration intobacteria and reduce drug bioactivity. The strategy of increasing drugdosing to overcome these obstacles is limited by drug toxicity. Toassess the impact of sputum on the antibacterial activity of P₄₀₇5EC,standard planktonic susceptibility testing was repeated for the 12biofilm-forming strains in the presence of CF sputum. A mixture ofsputum from 15 CF patients was used to avoid inter-patient variation inmacromolecule and high molecular weight DNA composition, and ionicconditions, and only mechanical shearing was applied to minimize changesto the native microenvironment (See, e.g., Grebski et al., Chest. 2001May; 119(5): 1521-5). The activity of P₄₀₇5EC against bacteria suspendedin media containing 43% sputum (the maximum sputum concentrationachieved in the test system) was decreased with bactericidalconcentrations 2- to 32-fold greater than the respective planktonic MBCswithout sputum. The sputum-MBCs were identical to (6 of 12) or withinone dilution of (6 of 12) the MBECs obtained with biofilm grownbacteria. Although the activity of P₄₀₇5EC was similarly antagonized byboth CF sputum and biofilm growth, it remained bactericidal for all thestrains tested under both test conditions (See FIG. 10).

Thus, in some embodiments, the present invention provides compositionscomprising nanoemulsions (e.g., P₄₀₇5EC) and methods of using the samefor antimicrobial treatment for infection due to CF-relatedopportunistic pathogens. In particular, nanoemulsion compositions of thepresent invention are rapidly bactericidal, and active against bacteriawhether grown planktonically or as a biofilm, or in the presence of CFsputum. Moreover, compositions comprising nanoemulsions of the presentinvention are exceptionally stable, unchanged after nebulization, andbroadly microbicidal. Importantly, the development of resistance to ananemulsion composition of the present invention has not been observedby any bacterial species examined to date. Thus, the present inventionprovides that P₄₀₇5EC can be used effectively as an inhaledantimicrobial.

The present invention is not limited to treatment of bacterial biofilmsthe reside in pulmonary spaces (e.g., within a subject with CF). Indeed,compositions comprising a nanoemulsion of the present invention can beutilized as a therapeutic and/or antimicrobial agent (e.g., to killand/or inhibit growth of) bacterial biofilms in any clinical and/orindustrial setting.

Multiple species of bacteria exist that are able to form biofilms. Forexample, bacteria that adhere to implanted medical devices or damagedtissue often encase themselves in a hydrated matrix of polysaccharideand protein to form biofilm. Biofilms pose a serious problem for publichealth because of the increased resistance of biofilm-associatedorganisms to antimicrobial agents and the association of infections withthese organisms in patients with indwelling medical devices or damagedtissue. Antibiotic resistance of bacteria growing in biofilmscontributes to the persistence and chronic nature of infections such asthose associated with implanted medical devices. The mechanisms ofresistance in biofilms are different from the now familiar plasmids,transposons, and mutations that confer innate resistance to individualbacterial cells. In biofilms, resistance seems to depend onmulticellular strategies.

Biofilms are complex communities of microorganisms attached to surfacesor associated with interfaces or damaged tissue. Despite the focus ofmodern microbiology research on pure culture, planktonic (free-swimming)bacteria, it is now widely recognized that most bacteria found innatural, clinical, and industrial settings persist in association withsurfaces as biofilms. Furthermore, these microbial communities are oftencomposed of multiple species that interact with each other and theirenvironment. The determination of biofilm architecture, particularly thespatial arrangement of microcolonies (clusters of cells) relative to oneanother, has profound implications for the function of these complexcommunities.

The biofilm matrix is a dynamic environment in which the componentmicrobial cells appear to reach homeostasis and are optimally organizedto make use of all available nutrients. The matrix therefore shows greatmicroheterogeneity, within which numerous microenvironments can exist.Biofilm formation is believed to be a two-step process in which theattachment of bacterial cells to a surface is followed by growthdependent accumulation of bacteria in multilayered cell clusters.Although exopolysaccharides provide the matrix framework, a wide rangeof enzyme activities can be found within the biofilm, some of whichgreatly affect structural integrity and stability.

More specifically, during the first phase of formation, it ishypothesized that the fibrinogen and fibronectin of host plasma coverthe surface of a medical implant or damaged tissue and are identified byconstitutively expressed microbial surface components, which mediate theinitial attachment of bacteria to the surface of the biomaterial ordamaged tissue. In the second step, a specific gene locus in thebacteria cells, called the intracellular adhesion (ica) locus, activatesthe adhesion of bacteria cells to each other, forming the secondarylayers of the biofilm. The ica locus is responsible for the expressionof the capsular polysaccharide operon, which in turn activatespolysaccharide intercellular adhesion (PIA), via the sugarpoly-N-succinylglucosamine (PNSG), a-1,6-linked glucosaminoglycan. Theproduction of this polysaccharide layer gives the biofilm its slimyappearance when viewed using electron microscopy.

Staphylococcus aureus is a highly virulent human pathogen. Both S.aureus and coagulase-negative staphylococci have emerged as majornosocomial pathogens associated with biofilm formation on implantedmedical devices and damaged tissue. These organisms are among the normalcarriage flora of human skin and mucous membranes, making them prevalentcomplications during and after invasive surgery or prolonged hospitalstays. As bacteria carried on both healthy and sick people,staphylococci are considered opportunistic pathogens that invadepatients via open wounds and via biomaterial implants.

Biofilm infections associated with S. aureus are a significant cause ofmorbidity and mortality, particularly in settings such as hospitals,nursing homes and infirmaries. Patients at risk include infants, theelderly, the immuno-compromised, the immuno-suppressed, and those withchronic conditions requiring frequent hospital stays. Patients withintravascular and other implanted prosthetic devices are at even greaterrisk from staphylococcal infections because of compromised immunesystems and the introduction of foreign bodies, which serve to damagetissue and/or act as a surface for the formation of biofilms. Suchinfections can have chronic, if not fatal, implications.

The causes of biofilm resistance to antibiotics include the failure ofsome antimicrobial agents to penetrate all the layers of a biofilm, theslow-growth rate of certain biofilm cells that make them lesssusceptible to antimicrobial agents requiring active bacterial growth,and the expression of gene patterns by the bacterial cells embedded inthe biofilm that differ from the genes expressed in their planktonic(free-swimming) state. These differences in biofilm-associated bacteriarender antimicrobial agents that work effectively to kill planktonicbacteria ineffective in killing biofilm-associated bacteria. Often theonly way to treat biofilms (e.g., associated with catheters orprosthetic devices) is the removal of the contaminated device, which mayrequire additional surgery and present further risks to patients.

Thus, as used herein, biofilms refer to an aggregate of microorganismswith an extracellular matrix that facilitates adhesion to, andcolonization and growth of the aggregate on a surface, such as aninternal or external tissue or organ. Biofilms can be comprised ofbacteria, fungi, yeast, protozoa, or other microorganisms. Bacterialbiofilms typically display high resistance to antibiotics, often up to1,000-times greater resistance than the same bacteria not growing in abiofilm.

In some embodiments, compositions and methods of the invention areutilized to treat (e.g., kill and/or inhibit growth of) and/or preventbiofilms on and/or within a subject (e.g., within the pulmonary system,on internal organs or tissue (e.g., the bladder, kidney, heart, middleear, sinuses, a joint, the eye), on an external tissue (e.g., the skin),and/or oral surfaces such as teeth, tongue, oral mucosa, or gums.Compositions and methods of the invention may be used to treat abiofilm-associated condition such as a soft-tissue infection, chronicsinusitis, endocarditis, osteomyelitis, urinary tract infection, chronicbacterial vaginosis, dental plaque or halitosis, infection of prostheticdevice and/or catheter, bacterial keratitis, or prostatitis.

As described in Examples 3 and 4, compositions of the present inventioncan be utilized to treat (e.g., kill and/or inhibit growth of) any oneor more Gram-positive and Gram-negative bacterial species. Indeed,compositions and methods of the present invention can be utilized tokill and/or inhibit growth of a number of bacterial species including,but not limited to, Staphylococcus aureus, coagulase negativestaphylococci such as Staphylococcus epidermis, Streptococcus pyogenes(Group A), Streptococcus species (viridans group), Streptococcusagalactiae (group B), S. bovis, Streptococcus (anaerobic species),Streptococcus pneumoniae, Enterococcus species, Bacillus anthracis,Corynebacterium diptheriae, and Corynebacterium species which arediptheroids (aerobic and anaerobic), Listeria monocytogenes, Clostridiumtetani, and Clostridium difficile, Escherichia coli, Enterobacterspecies, Proteus mirablis, Pseudomonas aeruginosa, Klebsiellapneumoniae, Salmonella, Shigella, Serratia, Campylobacterjejuni,Neisseria, Branhamella catarrhalis, and Pasteurella.

Compositions and methods of the present invention can be utilized totreat (e.g., kill and/or inhibit growth of) organisms capable of formingbiofilms including, but not limited to, dermatophytes (e.g, Microsporumspecies such as Microsporum canis, Trichophyton species such asTrichophyton rubrum and Trichophyton mentagrophytes), yeasts (e.g.,Candida albicans, Candidaparapsilosis, Candida glabrata, Candidatropicalis, and other Candida species including drug resistant Candidaspecies), Epidermophytonfloccosum, Malasseziafuurfur (Pityropsporonorbiculare, Pityropsporon ovale) Cryptococcus neoformans,Aspergillusfumigatus and other Aspergillus species, Zygomycetes(Rizopus, Mucor), hyalohyphomycosis (Fusarium species), Paracoccidioidesbrasiliensis, Blastmyces dermatitides, Histoplasma capsulatum,Coccidiodes immitis, Sporothrix schenckii, and Blastomyces.

Thus, in some embodiments, the present invention provides a method fortreating a subject possessing a biofilm (e.g., possessing an indwellingprosthetic device or catheter, wherein the indwelling prosthetic deviceor catheter is in contact with a biofilm, or wherein the subject has aninfection (e.g., respiratory infection) within which a biofilm resides)comprising administering to the subject a composition comprising ananoemulsion (e.g., P₄₀₇5EC) under conditions such that the biofilm isaltered and/or bacteria residing within the biofilm are killed and/ortheir growth is inhibited. In some embodiments, altering the biofilmcomprises eradicating the biofilm. In some embodiments, altering thebiofilm comprises killing bacteria involved in forming the biofilm. Insome embodiments, the bacteria comprise S. aureus, S. epidermidis,antibiotic resistant bacteria (e.g., methicillin resistant, vancomycinresistant, etc.), and/or other type of bacteria described herein. Insome embodiments, the composition comprising a nanoemulsion (e.g.,P₄₀₇5EC) is co-administered with one or more antibacterial agents. Insome embodiments, the antibacterial agents are selected from the groupcomprising, but not limited to, antibiotics, antibodies, antibacterialenzymes, peptides, and lanthione-containing molecules. In someembodiments, the antibiotic interferes with or inhibits cell wallsynthesis. In some embodiments, the antibiotic is selected from thegroup including, but not limited to, β-lactams, cephalosporins,glycopeptides, aminoglycosides, sulfonomides, macrolides, folates,polypeptides and combinations thereof. In some embodiments, theantibiotic interferes with protein synthesis (e.g., glycosides,tetracyclines and streptogramins). The present invention is not limitedby the number of doses of composition comprising nanoemulsionadministered. In some embodiments, multiple doses are administered onseparate days. In some embodiments, the multiple doses are administeredon the same day. In some embodiments, a composition comprising ananoemulsion described herein is administered continuously. In someembodiments, co-administration with a composition comprising ananoemulsion permits administering a lower dose of an antibacterialagent than would be administered without co-administration of acomposition comprising a nanoemulsion. In some embodiments, thecomposition comprising a nanoemulsion described herein is administeredusing a nebulizer. In some embodiments, administration isintramuscularly, subcutaneously, locally, directly into an infectedsite, directly onto an indwelling prosthetic device (e.g., a shunt,stent, scaffold for tissue construction, feeding tube, punctual plug,artificial joint, pacemaker, artificial valve, etc.) or catheter. Insome embodiments, administration is directly through a catheter.

The present invention is not limited by the type of microbe treated.Indeed a variety of microbial pathogens can be treated (e.g, killed(e.g., completely killed)) and/or the growth thereof prevented and/orattenuated in a subject using the compositions and methods of thepresent invention including, but not limited to, bacteria, viruses, andfungi described herein.

The present invention also provides compositions and methods fortreating (e.g., killing and/or inhibiting growth of) organisms thatheretofore display resistance to a broad spectrum of antibiotics (e.g.,species of the genus Acinetobacter).

Acinetobacter species are generally considered nonpathogenic to healthyindividuals. However, several species persist in hospital environmentsand cause severe, life-threatening infections in compromised patients(See, e.g., Gerischer U (editor). (2008). Acinetobacter MolecularBiology, 1st ed., Caister Academic Press). The spectrum of antibioticresistances of these organisms together with their survival capabilitiesmake them a threat to hospitals as documented by recurring outbreaksboth in highly developed countries and elsewhere. Infections occur inimmunocompromised individuals, and the strain A. baumannii is the secondmost commonly isolated nonfermenting bacteria in human specimens.Acinetobacter is frequently isolated in nosocomial infections and isespecially prevalent in intensive care units, where both sporadic casesas well as epidemic and endemic occurrence is common. A. baumannii is afrequent cause of nosocomial pneumonia, especially of late-onsetventilator associated pneumonia. It can cause various other infectionsincluding skin and wound infections, bacteremia, and meningitis. A.lwoffi is also causative of meningitis. A. baumannii can survive on thehuman skin or dry surfaces for weeks.

Since the start of the Iraq War, over 700 U.S. soldiers have beeninfected or colonized by A. baumannii. Four civilians undergoingtreatment for serious illnesses at Walter Reed Army Medical Center inWashington, D.C., contracted A. baumannii infections and died. AtLandstuhl Regional Medical Center, a U.S. military hospital in Germany,another civilian under treatment, a 63-year-old German woman, contractedthe same strain of A. baumannii infecting troops in the facility andalso died.

Acinetobacter species are innately resistant to many classes ofantibiotics, including penicillin, chloramphenicol, and oftenaminoglycosides. Resistance to fluoroquinolones has been reported duringtherapy and this has also resulted in increased resistance to other drugclasses mediated through active drug efflux. A dramatic increase inantibiotic resistance in Acinetobacter strains has been reported by theCDC and the carbapenems are recognized as the gold-standard and/ortreatment of last resort. An increase in resistance to the carbapenemsleaves very little treatment option although there has been some successreported with polymyxin B. Acinetobacter species are unusual in thatthey are sensitive to sulbactam; sulbactam is most commonly used toinhibit bacterial beta-lactamase, but this is an example of theantibacterial property of sulbactam itself.

Thus, in some embodiments, compositions and methods of the presentinvention are utilized to treat (e.g., kill and/or inhibit growth of)bacteria of the Acinetobacter species (e.g., individually or incombination with other treatments (e.g., carbapenems, polymyxin B,and/or sulbactam)).

Cystic Fibrosis

Cystic fibrosis (CF) is a life-threatening disorder that causes severelung damage due to a defective transmembrane protein called CFTRresponsible for the balance of electrolytes. Thick mucus forms pluggingthe tubes, ducts and passageways in the lungs. This environment is idealfor opportunistic bacteria to establish biofilm communities, leading torespiratory infections. Systemically-administered antibiotics candecrease the frequency and severity of exacerbations; however, thebacteria are never be completely eradicated from the airways and thelungs. Nebulized antibiotics are used, but resistance emergence and/orcolonization of different resistant species is a major concern. Cysticfibrosis (CF) results in the functional impairment of innate respiratorydefense mechanisms, providing an environment for colonization ofpathogenic bacterial species such as Staphylococcus aureus andHaemophilus influenzae, and a number of opportunistic species such asPseudomonas aeruginosa, Achromobacter xylosoxidans, Stenotrophomonasmaltophilia, Ralstonia spp., Pandoraea spp., and the Burkholderiacepacia complex (Bcc) species (See, e.g., LiPuma et al., (2009)Antimicrob Agents Chemother 53, 249-255). The Bcc comprises a group ofat least 17 phylogenetically related saprophytic gram-negative bacilli,most of which can form biofilm (See, e.g., Al Bakri et al., 2004,Journal of Applied Microbiology 96, 455-463; Eberl and Tummler, 2004,International Journal of Medical Microbiology 294, 123-131; LiPuma etal., (2009) Antimicrob Agents Chemother 53, 249-255; and Tomlin et al.,2004, Journal of Microbiological Methods 57, 95-106). They areparticularly difficult to treat and are associated with increased ratesof morbidity and mortality in CF patients. They also are among the mostantimicrobial-resistant bacterial species encountered in humaninfections (See, e.g., LiPuma et al, 2005, Curr Opin Pulm Med 11,528-533; LiPuma et al., (2009) Antimicrob Agents Chemother 53, 249-255).Once established, the infection and associated inflammation are rarelyeliminated, resulting in progressive lung disease ending in pulmonaryfailure and death (See, e.g., LiPuma et al., (2009) Antimicrob AgentsChemother 53, 249-255; Saiman and Seigel, 2003, Infect Control HospEpidemiol 24, S6-S52).

The present invention is directed to a novel broad-spectrumantimicrobial nanoemulsion (NE) and uses thereof. The NE kills pathogensby interacting with their membranes. This physical kill-on-contactmechanism significantly reduces any concerns about resistance. The NE isformulated from pharmaceutically approved safe ingredients.

In Example 8 below, the minimum inhibitory concentration (MIC) andminimum bactericidal concentration (MBC) of a against four genera ofproblematic bacteria in CF patients was evaluated: Pseudomonas,Burkholderia, Acinetobacter and Stenotrophomonas. Example 8 alsodescribes evaluating potential synergy between the NE and otherantimicrobials. P. aeruginosa, B. cenocepacia, A. baumannii and S.maltophilia, which were evaluated in Example 8, are importantrespiratory pathogens implicated in acute exacerbations of cysticfibrosis patients. In one aspect of the invention, the activity of ananoemulsion is defined herein in terms of its minimum inhibitoryconcentration (MIC), and/or minimum bactericidal concentration (MBC),both in comparison to traditionally used antimicrobial medicines

The results described in Example 8 show that the MIC₉₀/MBC₉₀ values forthe NE tested were 8/64 μg/ml for P. aeruginosa, 64/>514 μg/ml for B.cenocepacia, 8/64 μg/ml for A. baumannii and 8/32 μg/ml for S.maltophilia. Colistin had MIC₉₀/MBC₉₀ values of 2/8, >32/>32, 1/>16and >32/>32 for P. aeruginosa, B. cenocepacia, A. baumannii and S.maltophilia, respectively. Cefepime, imipenem, levofloxacin andtobramycin had MIC₉₀/MBC₉₀ values of ≧32/>32, ≧32/>32, 16/16 and >32/>32μg/ml, respectively, against all strains. These results are significantas in contrast to conventional drugs such as colistin, cefepime,imipenem, levofloxacin and tobramycin used to treat CF and which canexhibit significant side effects and potential toxicity with increasingdosage, NE are completely non-toxic.

Also evaluated in Example 8 below was synergy data. Specifically, alsoevaluated was the fractional inhibitory concentration (FIC) index whenin combination with another antimicrobial and the fractionalbactericidal concentration (FBC) index when in combination with anotherantimicrobial. The FIC and the FBC are calculated to make the judgmenton synergy, antagonism or indifference of the nanoemulsion incombination. For this determination, ten strains of Burkholderia,Stenotrophomonas and 10 strains of Acinetobacter were tested todetermine a shift in MIC when P₄₀₇5EC+EDTA was in combination witheither colistin or tobramycin, two traditional antimicrobials used inthe lungs of CF patients to treat chronic lung infections. The resultsshowed that the NE texted (P₄₀₇5EC+EDTA) in combination with colistinwas found to be synergistic for 90% (in terms of the FIC) and 70% (interms of the FBC) of the Stenotrophomonas strains, but indifferent, only20% synergy in by the FIC and 0% by the FBC, when in combination withtobramycin. For the Acinetobacter strains, P₄₀₇5EC+EDTA in combinationwith colistin was found to be indifferent, only 20% synergy in by theFIC and 0% by the FBC, as well as when in combination with tobramycin,only 10% synergy in by the FIC and 10% by the FBC. For the Burkholderiastrains, P₄₀₇5EC+EDTA in combination with colistin was found to beindifferent, only 30% synergy in by the FIC and 10% by the FBC, but whenin combination with tobramycin, 50% synergy in by the FIC and 20% by theFBC.

Finally, Example 8 generated a short time-kill curve to produce samplesto be used in pictures using electron microscopy for a strain ofBurkholderia to demonstrate the physical kill-on-contact mechanism ofaction. Time-kill resulted in an overall 4.44 log reduction in cfu/mlfrom the untreated beginning to the 30 minute time point. Each 10 minutetime point had between 1-2 log reduction as follows: from the untreatedto the 10 minute point there was a 1.40 log reduction, from the 10 to 20minute time points there was a 1.91 log reduction and from the 20 to 30minute time points there was a 1.13 log reduction. See FIGS. 2A-2D.

The data herein, such as that presented in Example 8, demonstrates thatnanoemulsions, such as the tested P₄₀₇5EC, are effective against strainsthat are multidrug-resistant, including colistin-resistant isolates ofBurkholderia and Stenotrophomonas. None of the described lipid Amodifications in Pseudomonas species impacted the MIC/MBCs with P₄₀₇5EC.No evidence of antagonism with two major antibiotics, colistin andtobramycin, was observed and in the case of the Stenotrophomonas,synergy was evident. This is valuable because the treatment of patientswith CF should not need their normal antibiotic regime suspended inorder to use the nanoemulsion, complicating their treatment programs.The SEM images demonstrate the kill on contact mechanism, here in thiscase a Gram negative bacterium with a reputation of having a tough outermembrane. Further studies are ongoing to investigate the nebulization ofP₄₀₇5EC for the treatment of pulmonary infection in cystic fibrosispatients.

While therapeutic progress has been made in preserving functionalpulmonary physiology in CF patients by managing nutrition and mucosalsecretions, little progress has been made in therapeutic interventionsfor the prevention and management of Bcc infections. For example, atpresent, no effective vaccines against Bcc are available. Mucosalvaccine development for Bcc has been limited in part due to the lack ofan identified protective antigen and the lack of effective mucosaladjuvants (See, e.g., Davis, 2001, Advanced Drug Delivery Reviews 51,21-42).

As shown in Example 10 below, the present invention provides immunogeniccompositions comprising a nanoemulsion and Burkholderia antigen (e.g.,Burkholderia outer membrane protein (OMP)) that, when administered to asubject, induces immunity (e.g., protective immunity) in the subjectagainst bacteria from the genus Burkholderia (e.g., B. cenocepacia, B.multivorans or others species associated with respiratory infection). Insome embodiments, the present invention provides all or a portion of anOMP (e.g., isolated, purified, and/or recombinant OMPs)) from a bacteriaof the genus Burkholderia (See, e.g., Example 10, Table 25) that isutilized in an immunogenic composition comprising a nanoemulsion (e.g.,for administration to a subject (e.g., to induce immunity to a bacteriaof the genus Burkholderia in the subject)). As shown in Example 10, thepresent invention provides that when administered to a subject, animmunogenic composition comprising a nanoemulsion and Burkholderia OMPinduces both mucosal and systemic anti-OMP antibodies. Also shown inExample 10, the present invention provides that when administered to asubject, an immunogenic composition comprising a nanoemulsion and an OMPfrom a specific strain of Burkholderia (e.g., B. cenocepacia) inducesboth mucosal and systemic anti-OMP antibodies that neutralize bacteriaof the specific species from which the OMP is derived (e.g., B.cenocepacia) and cross-neutralize bacteria from one or more otherspecies of bacteria (e.g., of the genus Burkholderia).

Example 10 shows that a single nasal immunization with OMP-NE produces arobust immune response characterized by rapid induction ofantigen-specific T cell responses and secretory antibody responses, asdocumented by induction of IFN-γ, IL-2 and mucosal sIgA and IgG, andthat the immune response can be subsequently boosted. Mice immunizedwith OMP-NE displayed a dramatic decrease in K56-1 colony forming units(cfu) in the lungs when compared with non-immunized controls. Pulmonarybacterial clearance was significantly enhanced in OMP-NE vaccinated micewhich showed fewer B. cenocepacia organisms in the spleens as comparedto non-vaccinated groups. Thus, the present invention provides thatmucosal OMP-NE administration produced protective immunity and reducedthe likelihood of sepsis in subjects.

Example 10 also provides data identifying a 17 KDa OmpA-like protein andimmune responses associated with exposure to the protein. The 17 KDalipoprotein fractions were better recognized by antibodies contained inserum from OMP-NE vaccinated mice (See, e.g., Example 10, FIG. 29C).Thus, the present invention provides that immunogenic compositionscomprising epitopes contained within the 17KDa OmpA-like proteinsgenerate protective antibodies (e.g., against one or more species ofBurkholderia) in a subject administered the immunogenic composition.

Significant differences between OMP-NE and OMP in PBS responses wereobserved. For example, immunization with OMP in the absence ofnanoemulsion resulted in relatively high titers of IgG, but wereconsistently lower (13 to 30 fold) than those induced by OMP-NEimmunogenic compositions. Immunization with OMP in the absence ofnanoemulsion failed to respond to a booster immunization. The responseinduced with OMP plus nanoemulsion was primarily directed towardspecific proteins contained within the preparation, whereas OMP in theabsence of nanoemulsion appears to have elicited immune responses to theintrinsic endotoxin content.

Accordingly, the present invention provides methods and compositions forthe stimulation of immune responses. In particular, the presentinvention provides immunogenic nanoemulsion compositions and methods ofusing the same for the induction of immune responses (e.g., innateand/or adaptive immune responses (e.g., for generation of host immunityagainst a bacterial species of the genus Burkholderia (e.g., B.cenocepacia, B. multivorans, etc.))). Compositions and methods of thepresent invention find use in, among other things, clinical (e.g.therapeutic and preventative medicine (e.g., vaccination)) and researchapplications.

In some embodiments, the present invention provides nanoemulsionadjuvants and compositions comprising the same (e.g., vaccines) for thestimulation of immune responses (e.g., immunity) against a bacterialspecies of the genus Burkholderia (e.g., B. cenocepacia, B. multivorans,etc.). In some embodiments, the present invention provides nanoemulsionadjuvant compositions that stimulate and/or elicit immune responses(e.g., innate immune responses and/or adaptive/acquired immuneresponses) when administered to a subject (e.g., a human or othermammalian subject)). In some embodiments, the present invention providesnanoemulsion adjuvant compositions comprising one or a plurality ofBurkholderia (e.g., B. cenocepacia, B. multivorans, etc.) antigens(e.g., Burkholderia components isolated and/or purified, and/orrecombinant Burkholderia proteins (e.g., OMPs)). The present inventionis not limited to any particular nanoemulsion or Burkholderia antigen.Exemplary immunogenic compositions (e.g., vaccine compositions) andmethods of administering the compositions are described in more detailbelow.

In some embodiments, the present invention provides an immunogeniccomposition comprising a nanoemulsion and one or more Burkholderiaantigens (e.g., B. cepacia antigens). In some embodiments, the presentinvention provides a method of inducing an immune response toBurkholderia (e.g., B. cepacia) in a subject comprising: providing asubject and an immunogenic composition comprising a nanoemulsion and animmunogen, wherein the immunogen comprises a Burkholderia (e.g.,Burkholderia cepacia) antigen and administering the composition to thesubject under conditions such that the subject generates a Burkholderia(e.g., Burkholderia cepacia) specific immune response. The presentinvention is not limited by the route chosen for administration of acomposition of the present invention. In some preferred embodiments,administering the immunogenic composition comprises contacting a mucosalsurface of the subject with the composition. In some embodiments, themucosal surface comprises nasal mucosa. In some embodiments, inducing animmune response induces immunity to Burkholderia (e.g., Burkholderiacepacia) in the subject.

Experiments were conducted during development of embodiments of theinvention to determine if a composition comprising a nanoemulsion (NE)and Burkholderia antigen could be utilized to generate an immuneresponse in a subject. Nasal immunization with a whole cellStreptococcus pneumoniae antigen (WCPAg) mixed with nanoemulsion wasperformed and shown to induce an IgG response in a host subject and theability to eradicate upper respiratory colonization of S. pneumoniae.

In particular, as described in Example 10, the present inventionprovides immunogenic compositions comprising a nanoemulsion andBurkholderia antigen (e.g., Burkholderia outer membrane protein (OMP))that, when administered to a subject, induces immunity (e.g., protectiveimmunity) in the subject against bacteria from the genus Burkholderia(e.g., B. cenocepacia, B. multivorans or others species associated withrespiratory infection). Accordingly, in some embodiments, the presentinvention provides that administration (e.g., nasal administration) of acomposition comprising nanoemulsion and Burkholderia antigen (e.g., OMPantigen (e.g., a 17KDa protein comprising an amino acid sequenceselected from SEQ ID NOs. 1-16)) to a subject produces immunity towardBurkholderia in the subject thereby protecting the subject againstBurkholderia infection (e.g., associated with a respiratory infection).

The present invention is not limited by the type of bacteria of thegenus Burkholderia utilized in the immunogenic compositions and methodsof using the same of the invention. In some embodiments, the bacteria isa pathogen. In some embodiments, the pathogen is a Burkholderia speciesresponsible for respiratory or respiratory associated infection. Avariety of Burkholderia species find use in the compositions and methodsof the invention including, but not limited to, B. cenocepacia, B.dolosa, B. multivorans, B. ambifaria, B. vietnamiensis, B. ubonensis, B.thailandensis, B. graminis, B. oklahomensis, B. pseudomallei, B.xenovorans, B. phytofirmans, B. phymatum, R. metallidurans, R. eutropha,R. solanacearum.

In some embodiments, the bacteria of the genus Burkholderia is B.cepecia. In some embodiments, an immunogenic composition comprising ananoemulsion and an Omp-A like protein comprises an Omp-A like proteinfrom a Burkholderia species including, but not limited to, B.cenocepacia, B. dolosa, B. multivorans, B. ambifaria, B. vietnamiensis,B. ubonensis, B. thailandensis, B. graminis, B. oklahomensis, B.pseudomallei, B. xenovorans, B. phytofirmans, B. phymatum, R.metallidurans, R. eutropha, R. solanacearum. In some embodiments, animmunogenic composition comprising a nanoemulsion and an Omp-A likeprotein comprises an Omp-A like protein (e.g., isolated, purified,and/or recombinant Omp-A like protein) comprising an amino acid sequenceidentified in SEQ ID NOs.: 1-16. In some embodiments, an immunogeniccomposition comprising a nanoemulsion and an Omp-A like proteincomprises an Omp-A like protein (e.g., isolated, purified, and/orrecombinant Omp-A like protein) comprising an amino acid sequence of SEQID NO. 1.

In some embodiments, an immunogenic composition comprising ananoemulsion and Burkholderia antigen comprises antigens (e.g.,polysaccharide, protein, killed whole cells (e.g., conjugated ornon-conjugated antigens)), wherein the antigens are derived frommultiple (e.g., at least 2, 3, 5, 7, 10, 15, 20 or more) serotypes ofBurkholderia. The number of Burkholderia antigens utilized can rangefrom 2 different serotypes to about 20 different serotypes. In someembodiments, an immunogenic composition comprising a nanoemulsion andBurkholderia antigen may comprise Burkholderia antigen (e.g., wholecell, polysaccharide, Omp-A protein, other protein, etc.) from everyknown and/or isolated Burkholderia serotype.

In some embodiments, an immunogenic composition comprising ananoemulsion and Burkholderia (e.g., B. cepacia) antigen comprises one,two or more different types of carrier protein (e.g., that act ascarriers for proteins, saccharides, etc.). For example, in oneembodiment, two or more different saccharides or proteins may beconjugated to the same carrier protein, either to the same molecule ofcarrier protein or to different molecules of the same carrier protein.Carrier proteins may be TT, DT, CRM197, fragment C of TT, PhtD, PhtBE orPhtDE fusions (particularly those described in WO 01/98334 and WO03/54007), detoxified pneumolysin and protein D. In some embodiments, acarrier protein present in a composition comprising a nanoemulsion andBurkholderia (e.g., B. cenocepacia) antigen is a member of thepolyhistidine triad family (Pht) proteins, fragments or fusion proteinsthereof. The PhtA, PhtB, PhtD or PhtE proteins may have an amino acidsequence sharing 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with asequence disclosed in WO 00/37105 or WO 00/39299 (e.g. with amino acidsequence 1-838 or 21-838 of SEQ ID NO: 4 of WO 00/37105 for PhtD). Forexample, fusion proteins are composed of full length or fragments of 2,3 or 4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins arePhtA/B, PhtA/D, PhtA/E, PhtB/A, PhtB/D, PhtB/E. PhtD/A. PhtD/B, PhtD/E,PhtE/A, PhtE/B and PhtE/D, wherein the proteins are linked with thefirst mentioned at the N-terminus (see for example WO01/98334). Carriersmay comprise histidine triad motif(s) and/or coiled coil regions. Ahistidine triad motif is the portion of polypeptide that has thesequence HxxHxH where H is histidine and x is an amino acid other thanhistidine. A coiled coil region is a region predicted by “Coils”algorithm Lupus, A et al (1991) Science 252; 1162-1164.

Examples of carrier proteins which may be used in the present inventionare DT (Diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, DTCRM197 (a DT mutant) other DT point mutants, such as CRM176, CRM228, CRM45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45,CRM102, CRM 103 and CRM107 and other mutations described by Nicholls andYoule in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc,1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S.Pat. No. 4,950,740; mutation of at least one or more residues Lys 516,Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S.Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed inU.S. Pat. No. 5,843,711, pneumococcal pneumolysin (Kuo et al (1995)Infect Immun 63; 2706-13) including ply detoxified in some fashion forexample dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX,including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins for examplePhtDE fusions, PhtBE fusions (WO 01/98334 and WO 03/54007), (Pht A-E aredescribed in more detail below) OMPC (meningococcal outer membraneprotein—usually extracted from N. meningitidis serogroup B—EP0372501),PorB (from N. meningitidis), PD (Haemophilus influenzae protein D—see,e.g., EP 0 594 610 B), or immunologically functional equivalentsthereof, synthetic peptides (EP0378881, EPO427347), heat shock proteins(WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EPO471177),cytokines, lymphokines, growth factors or hormones (WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (Falugi et al (2001) Eur J Immunol 31;3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72;4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptakeproteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761).

Generation of Antibodies

An immunogenic composition comprising a nanoemulsion and Burkholderia(e.g., B. cenocepacia) antigen can be used to immunize a mammal, such asa mouse, rat, rabbit, guinea pig, monkey, or human, to producepolyclonal antibodies. If desired, a Burkholderia (e.g., B. cenocepacia)antigen can be conjugated to a carrier protein, such as bovine serumalbumin, thyroglobulin, keyhole limpet hemocyanin or other carrierdescribed herein. Depending on the host species, various adjuvants canbe used to increase the immunological response. Such adjuvants include,but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminumhydroxide), and surface active substances (e.g. lysolecithin, pluronicpolyols, polyanions, peptides, nanoemulsions described herein, keyholelimpet hemocyanin, and dinitrophenol). Among adjuvants used in humans,BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especiallyuseful.

Monoclonal antibodies that specifically bind to a Burkholderia (e.g., B.cepacia) antigen can be prepared using any technique which provides forthe production of antibody molecules by continuous cell lines inculture. These techniques include, but are not limited to, the hybridomatechnique, the human B cell hybridoma technique, and the EBV hybridomatechnique (See, e.g., Kohler et al., Nature 256, 495 497, 1985; Kozboret al., J. Immunol. Methods 81, 3142, 1985; Cote et al., Proc. Natl.Acad. Sci. 80, 2026 2030, 1983; Cole et al., Mol. Cell. Biol. 62, 109120, 1984).

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (See, e.g., Morrison et al., Proc.Natl. Acad. Sci. 81, 68516855, 1984; Neuberger et al., Nature 312, 604608, 1984; Takeda et al., Nature 314, 452 454, 1985). Monoclonal andother antibodies also can be “humanized” to prevent a patient frommounting an immune response against the antibody when it is usedtherapeutically. Such antibodies may be sufficiently similar in sequenceto human antibodies to be used directly in therapy or may requirealteration of a few key residues. Sequence differences between rodentantibodies and human sequences can be minimized by replacing residueswhich differ from those in the human sequences by site directedmutagenesis of individual residues or by grating of entirecomplementarity determining regions.

Alternatively, humanized antibodies can be produced using recombinantmethods, as described below. Antibodies which specifically bind to aparticular antigen can contain antigen binding sites which are eitherpartially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to a particular antigen.Antibodies with related specificity, but of distinct idiotypiccomposition, can be generated by chain shuffling from randomcombinatorial immunoglobin libraries (See, e.g., Burton, Proc. Natl.Acad. Sci. 88, 11120 23, 1991).

Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(See, e.g., Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11).Single-chain antibodies can be mono- or bispecific, and can be bivalentor tetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught, for example, in Mallender & Voss,1994, J. Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (See, e.g., Verhaar etal., 1995, Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J.Immunol. Meth. 165, 81-91).

Antibodies which specifically bind to a particular antigen also can beproduced by inducing in vivo production in the lymphocyte population orby screening immunoglobulin libraries or panels of highly specificbinding reagents as disclosed in the literature (See, e.g., Orlandi etal., Proc. Natl. Acad. Sci. 86, 3833 3837, 1989; Winter et al., Nature349, 293 299, 1991).

Chimeric antibodies can be constructed as disclosed in WO 93/03151.Binding proteins which are derived from immunoglobulins and which aremultivalent and multispecific, such as the “diabodies” described in WO94/13804, also can be prepared. Antibodies can be purified by methodswell known in the art. For example, antibodies can be affinity purifiedby passage over a column to which the relevant antigen is bound. Thebound antibodies can then be eluted from the column using a buffer witha high salt concentration.

Burn Wound Management

Contemporary burn wound management involves early debridement andreconstruction of non-viable skin coupled with provision of supportivecare and topical antimicrobial dressing changes to partial thicknessburn wounds. The goal of modern burn wound care is to provide an optimalenvironment for epidermal renewal. Restoration of skin integrity takesplace via regrowth of keratinocytes from preserved hair follicles ortransfer of split thickness skin grafts harvested from non-burn regions.During the period of epidermal renewal it is important to avoid furtherinjury to the skin, abrogate burn wound progression, and minimizesecondary complications such as wound infection. Early excision offull-thickness burn eschar, immediate skin grafting, and treatment ofremaining open or partial thickness areas of burn wound with topicalantimicrobial agents is the most effective way of minimizing burn woundcolonization and invasive wound infection. (See, e.g., Bessey, Woundcare. In Herndon D N, ed: Total Burn Care 3^(rd) edition. Philadelphia,Pa.: Elsevier Inc., 2007, pp 127-135.). Popular topical antimicrobialagents include silver sulfadiazine (SILVADENE), mafenide acetate(SULFAMYLON), and colloidal silver impregnated dressings (ACTICOAT,SILVERLON). Each of these agents has potential limitations such asvariable ability to penetrate eschar, uneven efficacy against bothGram-negative and Gram-positive bacteria, and potential toxicity to hostimmune cells (See, e.g., Steinstraesser et al., Antimicrob AgentsChemother 46(6):1837-1844, 2002).

Accordingly, the present invention provides nanoemulsion compositionsand methods of using the same for the treatment of burn wounds. Forexample, as shown in Example 9, the present invention providesnanoemulsion compositions and methods of using the same to reduce,attenuate and/or prevent bacterial growth in a burn wound. The presentinvention also provides nanoemulsion compositions that reduce woundinflammation following burn injury. Although an understanding of amechanism of action is not needed to practice the present invention, andthe present invention is not limited to any particular mechanism ofaction, in some embodiments, a nanoemulsion composition that is appliedto a wound following burn injury is able to penetrate more deeply anduniformly into a burn wound (e.g., thereby decreasing and/or inhibitingbacterial growth and/or inflammation at the site of the wound). As shownin Example 9, the present invention provides a method of reducing,inhibiting and/or eliminating bacterial growth in a burn woundcomprising providing a burn wound and a nanoemulsion and administeringthe nanoemulsion to the burn wound under conditions that bacterialgrowth is reduced, inhibited and/or eliminated. In some embodiments, ananoemulsion composition described herein is combined with one or moreantimicrobial drugs for administration to a burn wound to minimizebacterial growth at the burn wound site. The present invention is notlimited to any particular antimicrobial drug. Indeed, any antimicrobialdrug that inhibits bacterial growth known to those in the art can beutilized in combination with a nanoemulsion composition describedherein.

In addition to local effects, severe dermal burns are known to inducethe systemic inflammatory response syndrome (SIRS), which results in ahigh-risk of end-organ dysfunction (See, e.g., Barton et al., J BurnCare Rehabil 18(1):1-9, 1997). Increased vascular permeability andsystemic capillary leak as a consequence of SIRS following burn injurycreates seepage of plasma into interstitial tissue throughout the body.This tissue edema and intravascular hypovolemia is responsible for ahost of undesired clinical problems such as shock, pulmonarydysfunction, abdominal or extremity compartment syndrome, and cardiacfailure.

As shown in Example 9 below, nanoemulsion compositions described hereincan be administered to a burn wound to treat (e.g., reduce, attenuateand/or prevent) inflammation, tissue edema and/or intravascularhypovolemia at the site of a burn wound. In some embodiments, reducinginflammation, tissue edema and/or intravascular hypovolemia at the siteof a burn wound reduces the occurrence of shock, pulmonary dysfunction,abdominal or extremity compartment syndrome, and/or cardiac failure. Insome embodiments, a nanoemulsion composition described herein is used incombination with (e.g., is co-administered with) one or moreanti-inflammatory drugs to minimize early burn wound inflammation andtissue edema. The present invention is not limited to any particularanti-inflammatory drug. Indeed, any anti-inflammatory drug thatminimizes early burn wound inflammation and tissue edema can be utilizedin combination with a nanoemulsion composition described herein.

In some embodiments, a nanoemulsion of the invention (e.g., W₂₀5GBA₂ED)is administered to a burn wound to prevent, attenuate and/or eradicatebacterial growth (e.g., Staphylococcus aureus, P. aeruginosa, or otherbacteria) within a partial thickness burn wound. Example 9 shows thatreduction in microbial infection was coupled with generation of lowerlevels of local dermal pro-inflammatory cytokines and evidence ofreduced neutrophil sequestration into the burn wound. This decrease inburn wound bacterial growth and inflammation also produced lesscapillary leak in the early post-thermal injury time-period. Having theability to clinically reduce capillary leak and tissue edema in theimmediate post-burn time-period provides a lesser need for large volumecrystalloid fluid resuscitation and a reduction in the associatedsequela of physiologic volume overload, pulmonary dysfunction, andabdominal compartment syndrome.

Skin that is damaged by thermal injury loses its ability to protect thehost against infection from both the loss of physical barrier functionand the secondary immunosuppression caused by the thermal injury.Moreover, increased production of TGF-β and IL-10 during the post-burnperiod can result in immunosuppression. (See, e.g., Lyons et al., ArchSurg 134(12):1317-1323, 1999; Varedi et al., Shock 16(5):380-382, 2001).It has been established that treatment of burn injured animals withanti-TGF-β can improve local and systemic clearance of P. aeruginosa(See, e.g., Huang et al., J Burn Care Res 27(5):682-687, 2006)Inhibition of TGF-β also results in increased survival followingbacterial challenge. As shown in Example 9, a significant elevation ofTGF-β, but not IL-10 was observed in the skin following partialthickness burn injury. However, topical nanoemulsion application (e.g.,10% W₂₀5GBA₂ED) to the burn wound inoculated with bacteria resulted in areduction of the level of TGF-β when compared to the untreated burnwound.

Onset of a bacterial infection within a burn wound can delay or evenreverse the tissue healing process (See, e.g., Steinstraesser et al.,Crit Care Med 29(7):1431-1437, 2001). Topical antimicrobial therapy isused to reduce the microbial load in the burn wound and reduce this riskof infection. Current topical agents include silver nitrate (AgNO₃),silver sulfadiazine, mafenide acetate, and nanocrystalline impregnatedsilver dressings. Silver nitrate is limited in its use because of theproblem it creates from contact staining and its limited antifungalactivity. Silver sulfadiazine is the mainstay of topical burnantimicrobial treatment. It is bactericidal against P. aeruginosa andother Gram-negative enteric bacteria. Resistance to Silvadene by some ofthese organisms has emerged (See, e.g., Silver et al., J Ind MicrobiolBiotechnol 33(7):627-634, 2006). The agent has limited antifungalactivity, but can be used in conjunction with nystatin. Silvadene has noreal ability to penetrate burn eschar and sometimes leads to leukopeniawhich requires conversion to another topical agent. The use of mafenideacetate is narrowed by the fact that it is bacteriostatic against selectorganisms, it has limited activity against Gram-positive bacteria suchas Staphylococcus aureus, and that its use over a large surface area canlead to a metabolic acidosis because of its metabolism into a carbonicanhydrase inhibitor. The nanocrystalline silver dressings have thebroadest activity against burn wound pathogens of the current agentsavailable. They have a modest ability to penetrate eschar and can beleft in place for many days (See, e.g., Church et al., Clin MicrobiolRev 19(2):403-434, 2006). SB 202190, an inhibitor of activated p38 MAPK,can substantially reduce the dermal inflammation generated in burnwounds (See, e.g., Arbabi et al., Shock. 26(2):201-209, 2006). Thermalinjury initiates dermal inflammatory and pro-apoptotic cell signaling.

As shown in Example 9, topical application of nanoemulsion (e.g.,W₂₀5GBA₂ED resulted in reduced hair follicle cell apoptosis within thedermis of burned skin. Thus, in some embodiments, the present inventionprovides nanoemulsion compositions that can be utilized to reduce, whenadministered to a burn wound, conversion of the partial thickness burnwound within the “zone of stasis” to regions of full thickness burn.

In patients without evidence of inhalational injury, the burn wounditself is the primary source triggering the systemic inflammatoryresponse via generation of pro-inflammatory cytokines and sequestrationof neutrophils into the burn wound (See, e.g., Hansbrough et al., J SurgRes 61(1):17-22, 1996; Piccolo et al., Inflammation 23(4):371-385, 1999;Till et al., J Clin Invest 69(5):1126-1135, 1982). Topical applicationof a p38 MAPK inhibitor can control the source of inflammation at thelevel of the dermis, resulting in lower levels of pro-inflammatorymediators, reduced neutrophil sequestration and microvascular damage,and less epithelial apoptosis in burn wound hair follicle cells (See,e.g., Ipaktchi et al., Shock. 26(2):201-209, 2006). Dermal sourcecontrol of inflammation also reduces bacterial growth and attenuates thesystemic inflammatory response resulting in less acute lung injury andcardiac dysfunction following partial thickness burn injury in a rodentmodel. Accordingly, in some embodiments, a nanoemulsion of the inventionis utilized (e.g., administered) alone or in combination with ananti-inflammatory and/or antimicrobial agent to reduce local dermalinflammation and risk of infection within burn wounds (e.g., early burnwounds, partial thickness wounds, full thickness wounds or other burnwounds). The present invention is not limited by the type ofanti-inflammatory agent and/or antimicrobial utilized forco-administration with a nanoemulsion described herein. Indeed, avariety of anti-inflammatory agents and/or antimicrobial agents can beused including, but not limited to, silver nitrate (AgNO₃), silversulfadiazine, mafenide acetate, nanocrystalline impregnated silverdressings, p38 MAPK inhibitor (e.g., SB 202190), or anotheranti-inflammatory or antimicrobial agent described herein.

In some embodiments, when a nanoemulsion of the invention isadministered to a burn wound, the nanoemulsion can be administered(e.g., to a subject (e.g., to a burn or wound surface)) by multiplemethods, including, but not limited to, direct use or being suspended ina solution (e.g., colloidal solution) and applied to a surface (e.g., asurface comprising bacteria (e.g., pathogenic bacteria) or susceptibleto bacterial invasion); being sprayed onto a surface using a sprayapplicator; being mixed with fibrin glue and applied (e.g., sprayed)onto a surface (e.g., skin burn or wound); being impregnated onto awound dressing or bandage and applying the bandage to a surface (e.g.,an infection or burn wound); being applied by a controlled-releasemechanism; or being impregnated on one or both sides of an acellularbiological matrix that is then placed on a surface (e.g., skin burn orwound) thereby protecting at both the wound and graft interfaces. Insome embodiments, the invention provides a pharmaceutical compositioncontaining (a) a composition comprising a nanoemulsion (e.g.,W₂₀5GBA₂ED); and (b) one or more other agents (e.g., an antibiotic).Examples of other types of antibiotics include, but are not limited to,almecillin, amdinocillin, amikacin, amoxicillin, amphomycin,amphotericin B, ampicillin, azacitidine, azaserine, azithromycin,azlocillin, aztreonam, bacampicillin, bacitracin, benzylpenicilloyl-polylysine, bleomycin, candicidin, capreomycin,carbenicillin, cefaclor, cefadroxil, cefamandole, cefazoline, cefdinir,cefepime, cefixime, cefinenoxime, cefinetazole, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin,cefpiramide, cefpodoxime, cefprozil, cefsulodin, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cephacetrile,cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin,cephradine, chloramphenicol, chlortetracycline, cilastatin, cinnamycin,ciprofloxacin, clarithromycin, clavulanic acid, clindamycin, clioquinol,cloxacillin, colistimethate, colistin, cyclacillin, cycloserine,cyclosporine, cyclo-(Leu-Pro), dactinomycin, dalbavancin, dalfopristin,daptomycin, daunorubicin, demeclocycline, detorubicin, dicloxacillin,dihydrostreptomycin, dirithromycin, doxorubicin, doxycycline,epirubicin, erythromycin, eveminomycin, floxacillin, fosfomycin, fusidicacid, gemifloxacin, gentamycin, gramicidin, griseofulvin, hetacillin,idarubicin, imipenem, iseganan, ivermectin, kanamycin, laspartomycin,linezolid, linocomycin, loracarbef, magainin, meclocycline, meropenem,methacycline, methicillin, mezlocillin, minocycline, mitomycin,moenomycin, moxalactam, moxifloxacin, mycophenolic acid, nafcillin,natamycin, neomycin, netilmicin, niphimycin, nitrofurantoin, novobiocin,oleandomycin, oritavancin, oxacillin, oxytetracycline, paromomycin,penicillamine, penicillin G, penicillin V, phenethicillin, piperacillin,plicamycin, polymyxin B, pristinamycin, quinupristin, rifabutin,rifampin, rifamycin, rolitetracycline, sisomicin, spectrinomycin,streptomycin, streptozocin, sulbactam, sultamicillin, tacrolimus,tazobactam, teicoplanin, telithromycin, tetracycline, ticarcillin,tigecycline, tobramycin, troleandomycin, tunicamycin, tyrthricin,vancomycin, vidarabine, viomycin, virginiamycin, BMS-284,756, L-749,345,ER-35,786, S-4661, L-786,392, MC-02479, PepS, RP 59500, and TD-6424. Insome embodiments, two or more combined agents (e.g., a compositioncomprising a nanoemulsion and an antibiotic) may be used together orsequentially. In some embodiments, an antibiotic may comprisebacteriocins, type A lantibiotics, type B lantibiotics, liposidomycins,mureidomycins, alanoylcholines, quinolines, eveminomycins,glycylcyclines, carbapenems, cephalosporins, streptogramins,oxazolidonones, tetracyclines, cyclothialidines, bioxalomycins, cationicpeptides, and/or protegrins. In some embodiments, the compositioncomprises lysostaphin.

The present invention is not limited by the type of nanoemulsionutilized (e.g., for respiratory administration, administration to a burnwound and/or for use in an immunogenic composition for induction ofprotective immune responses). Indeed, a variety of nanoemulsioncompositions are contemplated to be useful in the present invention.

For example, in some embodiments, a nanoemulsion comprises (i) anaqueous phase; (ii) an oil phase; and at least one additional compound.In some embodiments of the present invention, these additional compoundsare admixed into either the aqueous or oil phases of the composition. Inother embodiments, these additional compounds are admixed into acomposition of previously emulsified oil and aqueous phases. In certainof these embodiments, one or more additional compounds are admixed intoan existing emulsion composition immediately prior to its use. In otherembodiments, one or more additional compounds are admixed into anexisting emulsion composition prior to the compositions immediate use.

Additional compounds suitable for use in a nanoemulsion of the presentinvention include, but are not limited to, one or more organic, and moreparticularly, organic phosphate based solvents, surfactants anddetergents, cationic halogen containing compounds, germinationenhancers, interaction enhancers, food additives (e.g., flavorings,sweeteners, bulking agents, and the like) and pharmaceuticallyacceptable compounds. Certain exemplary embodiments of the variouscompounds contemplated for use in the compositions of the presentinvention are presented below. Unless described otherwise, nanoemulsionsare described in undiluted form.

Stability on Storage and after Application of the Nanoemulsions of theInvention

Storage Stability

The nanoemulsions of the invention can be stable at about 40° C. andabout 75% relative humidity for a time period of at least up to about 1month, at least up to about 3 months, at least up to about 6 months, atleast up to about 12 months, at least up to about 18 months, at least upto about 2 years, at least up to about 2.5 years, or at least up toabout 3 years.

In another embodiment of the invention, the nanoemulsions of theinvention can be stable at about 25° C. and about 60% relative humidityfor a time period of at least up to about 1 month, at least up to about3 months, at least up to about 6 months, at least up to about 12 months,at least up to about 18 months, at least up to about 2 years, at leastup to about 2.5 years, or at least up to about 3 years, at least up toabout 3.5 years, at least up to about 4 years, at least up to about 4.5years, or at least up to about 5 years.

Further, the nanoemulsions of the invention can be stable at about 4° C.for a time period of at least up to about 1 month, at least up to about3 months, at least up to about 6 months, at least up to about 12 months,at least up to about 18 months, at least up to about 2 years, at leastup to about 2.5 years, at least up to about 3 years, at least up toabout 3.5 years, at least up to about 4 years, at least up to about 4.5years, at least up to about 5 years, at least up to about 5.5 years, atleast up to about 6 years, at least up to about 6.5 years, or at leastup to about 7 years.

Stability Upon Application

The nanoemulsions of the invention are stable upon application, assurprisingly the nanoemulsions do not lose their physical structure uponapplication. Microscopic examination of skin surface followingapplication of a nanoemulsion according to the invention demonstratesthe physical integrity of the nanoemulsions of the invention. Thisphysical integrity may result in the desired absorption observed withthe nanoemulsions of the invention.

Nanoemulsions

The term “nanoemulsion”, as defined herein, refers to a dispersion ordroplet or any other lipid structure. Typical lipid structurescontemplated in the invention include, but are not limited to,unilamellar, paucilamellar and multilamellar lipid vesicles, micellesand lamellar phases.

The nanoemulsion of the present invention comprises droplets having anaverage diameter size of less than about 1,000 nm, less than about 950nm, less than about 900 nm, less than about 850 nm, less than about 800nm, less than about 750 nm, less than about 700 nm, less than about 650nm, less than about 600 nm, less than about 550 nm, less than about 500nm, less than about 450 nm, less than about 400 nm, less than about 350nm, less than about 300 nm, less than about 250 nm, less than about 200nm, less than about 150 nm, or any combination thereof. In oneembodiment, the droplets have an average diameter size greater thanabout 125 nm and less than or equal to about 300 nm. In a differentembodiment, the droplets have an average diameter size greater thanabout 50 nm or greater than about 70 nm, and less than or equal to about125 nm. In other embodiments of the invention, the nanoemulsion dropletshave an average diameter of from about 300 nm to about 600 nm; or thenanoemulsion droplets have an average diameter of from about 150 nm toabout 400 nm.

In one embodiment of the invention, the nanoemulsion has a narrow rangeof MIC (minimum inhibitory concentration) and MBC (minimum bactericidalconcentrations) values. In another embodiment, the MIC and MBC for thenanoemulsion differ by less than or equal to four-fold, meaning that thenanoemulsion is bactericidal. In addition, the MIC and MBC for thenanoemulsion may differ by greater than four-fold, meaning that thenanoemulsion is bacteriostatic.

In one embodiment of the invention, the nanoemulsion comprises: (a) anaqueous phase; (b) about 1% oil to about 80% oil; (c) about 0.1% organicsolvent to about 50% organic solvent; (d) about 0.001% surfactant ordetergent to about 10% surfactant or detergent; (e) about 0.0005% toabout 1.0% of a chelating agent; or (e) any combination thereof. Inanother embodiment of the invention, the nanoemulsion comprises: (a)about 10% oil to about 80% oil; (b) about 1% organic solvent to about50% organic solvent; (c) at least one non-ionic surfactant present in anamount of about 0.1% to about 10%; (d) at least one cationic agentpresent in an amount of about 0.01% to about 3%; or any combinationthereof.

In another embodiment, the nanoemulsion comprises a cationic surfactantwhich is either cetylpyridinium chloride (CPC) or benzalkonium chloride,or alkyl dimethyl benzyl ammonium chloride (BTC 824), or combinationthereof. The cationic surfactant may have a concentration in thenanoemulsion of less than about 5.0% and greater than about 0.001%, orfurther, may have a concentration of less than about 5%, less than about4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%,less than about 2.5%, less than about 2.0%, less than about 1.5%, lessthan about 1.0%, less than about 0.90%, less than about 0.80%, less thanabout 0.70%, less than about 0.60%, less than about 0.50%, less thanabout 0.40%, less than about 0.30%, less than about 0.20%, less thanabout 0.10%, greater than about 0.001%, greater than about 0.002%,greater than about 0.003%, greater than about 0.004%, greater than about0.005%, greater than about 0.006%, greater than about 0.007%, greaterthan about 0.008%, greater than about 0.009%, and greater than about0.010%.

In a further embodiment, the nanoemulsion comprises a non-ionicsurfactant, and may have a concentration of about 0.01% to about 10.0%,or about 0.1% to about 3% of a non-ionic surfactant, such as apolysorbate.

In yet other embodiments of the invention, the nanoemulsion: (a)comprises at least one cationic surfactant; (b) comprises a cationicsurfactant which is either cetylpyridinium chloride or benzalkoniumchloride, or alkyl dimethyl benzyl ammonium chloride (BTC 824), orcombination thereof; (c) comprises a cationic surfactant, and whereinthe concentration of the cationic surfactant is less than about 5.0% andgreater than about 0.001%; (d) comprises a cationic surfactant, andwherein the concentration of the cationic surfactant is selected fromthe group consisting of less than about 5%, less than about 4.5%, lessthan about 4.0%, less than about 3.5%, less than about 3.0%, less thanabout 2.5%, less than about 2.0%, less than about 1.5%, less than about1.0%, less than about 0.90%, less than about 0.80%, less than about0.70%, less than about 0.60%, less than about 0.50%, less than about0.40%, less than about 0.30%, less than about 0.20%, less than about0.10%, greater than about 0.001%, greater than about 0.002%, greaterthan about 0.003%, greater than about 0.004%, greater than about 0.005%,greater than about 0.006%, greater than about 0.007%, greater than about0.008%, greater than about 0.009%, and greater than about 0.010%; or (e)any combination thereof. In yet other embodiments, (a) the nanoemulsioncomprises at least one cationic surfactant and at least one non-cationicsurfactant; (b) the nanoemulsion comprises at least one cationicsurfactant and at least one non-cationic surfactant, wherein thenon-cationic surfactant is a nonionic surfactant; (c) the nanoemulsioncomprises at least one cationic surfactant and at least one non-cationicsurfactant, wherein the non-cationic surfactant is a polysorbatenonionic surfactant; (d) the nanoemulsion comprises at least onecationic surfactant and at least one non-cationic surfactant, whereinthe non-cationic surfactant is a nonionic surfactant, and the non-ionicsurfactant is present in a concentration of about 0.05% to about 10%,about 0.05% to about 7.0%, about 0.1% to about 7%, or about 0.5% toabout 5%; (e) the nanoemulsion comprises at least one cationicsurfactant and at least one a nonionic surfactant, wherein the cationicsurfactant is present in a concentration of about 0.05% to about 2% orabout 0.01% to about 2%; or (f) any combination thereof.

In other embodiments, the nanoemulsion comprises: (a) water; (b) ethanolor glycerol (glycerine), or a combination thereof; (c) eithercetylpyridinium chloride (CPC), or benzalkonium chloride, or alkyldimethyl benzyl ammonium chloride (BTC 824), or a combination thereof;(c) soybean oil; and (e) Poloxamer 407, Tween 80, or Tween 20. Thenanoemulsion can further comprise EDTA.

These quantities of each component present in the nanoemulsion refer toa therapeutic nanoemulsion, and not to a nanoemulsion to be tested invitro. This is significant, as nanoemulsions tested in vitro generallyhave lower concentrations of oil, organic solvent, surfactant ordetergent, and (if present) chelating agent than that present in ananoemulsion intended for therapeutic use, e.g., topical use. This isbecause in vitro studies do not require the nanoemulsion droplets totraverse the skin. For topical, aerosol, intradermal etc. use, theconcentrations of the components must be higher to result in atherapeutic nanoemulsion.

However, the relative quantities of each component used in ananoemulsion tested in vitro are applicable to a nanoemulsion to be usedtherapeutically and, therefore, in vitro quantities can be scaled up toprepare a therapeutic composition, and in vitro data is predictive oftopical application success.

1. Aqueous Phase

The aqueous phase can comprise any type of aqueous phase including, butnot limited to, water (e.g., H₂O, distilled water, tap water) andsolutions (e.g., phosphate buffered saline (PBS) solution). In certainembodiments, the aqueous phase comprises water at a pH of about 4 to 10,preferably about 6 to 8. The water can be deionized (hereinafter“DiH₂O”). In some embodiments the aqueous phase comprises phosphatebuffered saline (PBS). The aqueous phase may further be sterile andpyrogen free.

2. Organic Solvents

Organic solvents in the nanoemulsions of the invention include, but arenot limited to, C₁-C₁₂ alcohol, diol, triol, dialkyl phosphate,tri-alkyl phosphate, such as tri-n-butyl phosphate, semi-syntheticderivatives thereof, and combinations thereof. In one aspect of theinvention, the organic solvent is an alcohol chosen from a nonpolarsolvent, a polar solvent, a protic solvent, or an aprotic solvent.

Suitable organic solvents for the nanoemulsion include, but are notlimited to, ethanol, methanol, isopropyl alcohol, glycerol, medium chaintriglycerides, diethyl ether, ethyl acetate, acetone, dimethyl sulfoxide(DMSO), acetic acid, n-butanol, butylene glycol, perfumers alcohols,isopropanol, n-propanol, formic acid, propylene glycols, glycerol,sorbitol, industrial methylated spirit, triacetin, hexane, benzene,toluene, diethyl ether, chloroform, 1,4-dixoane, tetrahydrofuran,dichloromethane, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, formic acid, semi-synthetic derivatives thereof, and anycombination thereof.

3. Oil Phase

The oil in the nanoemulsion of the invention can be any cosmetically orpharmaceutically acceptable oil. The oil can be volatile ornon-volatile, and may be chosen from animal oil, vegetable oil, naturaloil, synthetic oil, hydrocarbon oils, silicone oils, semi-syntheticderivatives thereof, and combinations thereof.

Suitable oils include, but are not limited to, mineral oil, squaleneoil, flavor oils, silicon oil, essential oils, water insoluble vitamins,Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate,Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthylanthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate,neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyladipate, C₁₂₋₁₅ alkyl lactates, Cetyl lactate, Lauryl lactate,Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate,Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluidparaffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil,Coconut oil, corn oil, Cottonseed oil, Flaxseed oil, Grape seed oil,Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seedoil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Teaoil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil(simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheatgerm oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil,Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nutoil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniperoil, seed oil, almond seed oil, anise seed oil, celery seed oil, cuminseed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil,cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemongrass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leafoil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmintleaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil,flower oil, chamomile oil, clary sage oil, clove oil, geranium floweroil, hyssop flower oil, jasmine flower oil, lavender flower oil, manukaflower oil, Marhoram flower oil, orange flower oil, rose flower oil,ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil,sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewoodoil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincenseoil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemonpeel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil,valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearylalcohol, semi-synthetic derivatives thereof, and any combinationsthereof.

The oil may further comprise a silicone component, such as a volatilesilicone component, which can be the sole oil in the silicone componentor can be combined with other silicone and non-silicone, volatile andnon-volatile oils. Suitable silicone components include, but are notlimited to, methylphenylpolysiloxane, simethicone, dimethicone,phenyltrimethicone (or an organomodified version thereof), alkylatedderivatives of polymeric silicones, cetyl dimethicone, lauryltrimethicone, hydroxylated derivatives of polymeric silicones, such asdimethiconol, volatile silicone oils, cyclic and linear silicones,cyclomethicone, derivatives of cyclomethicone,hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane,decamethylcyclopentasiloxane, volatile linear dimethylpolysiloxanes,isohexadecane, isoeicosane, isotetracosane, polyisobutene, isooctane,isododecane, semi-synthetic derivatives thereof, and combinationsthereof.

The volatile oil can be the organic solvent, or the volatile oil can bepresent in addition to an organic solvent. Suitable volatile oilsinclude, but are not limited to, a terpene, monoterpene, sesquiterpene,carminative, azulene, menthol, camphor, thujone, thymol, nerol,linalool, limonene, geraniol, perillyl alcohol, nerolidol, farnesol,ylangene, bisabolol, farnesene, ascaridole, chenopodium oil,citronellal, citral, citronellol, chamazulene, yarrow, guaiazulene,chamomile, semi-synthetic derivatives, or combinations thereof

In one aspect of the invention, the volatile oil in the siliconecomponent is different than the oil in the oil phase.

4. Surfactants/Detergents

The surfactant or detergent in the nanoemulsion of the invention can bea pharmaceutically acceptable ionic surfactant, a pharmaceuticallyacceptable nonionic surfactant, a pharmaceutically acceptable cationicsurfactant, a pharmaceutically acceptable anionic surfactant, or apharmaceutically acceptable zwitterionic surfactant.

Exemplary useful surfactants are described in Applied Surfactants:Principles and Applications. Tharwat F. Tadros, Copyright 8 2005WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30629-3), whichis specifically incorporated by reference.

Further, the surfactant can be a pharmaceutically acceptable ionicpolymeric surfactant, a pharmaceutically acceptable nonionic polymericsurfactant, a pharmaceutically acceptable cationic polymeric surfactant,a pharmaceutically acceptable anionic polymeric surfactant, or apharmaceutically acceptable zwitterionic polymeric surfactant. Examplesof polymeric surfactants include, but are not limited to, a graftcopolymer of a poly(methyl methacrylate) backbone with multiple (atleast one) polyethylene oxide (PEO) side chain, polyhydroxystearic acid,an alkoxylated alkyl phenol formaldehyde condensate, a polyalkyleneglycol modified polyester with fatty acid hydrophobes, a polyester,semi-synthetic derivatives thereof, or combinations thereof.

Surface active agents or surfactants, are amphipathic molecules thatconsist of a nonpolar hydrophobic portion, usually a straight orbranched hydrocarbon or fluorocarbon chain containing 8-18 carbon atoms,attached to a polar or ionic hydrophilic portion. The hydrophilicportion can be nonionic, ionic or zwitterionic. The hydrocarbon chaininteracts weakly with the water molecules in an aqueous environment,whereas the polar or ionic head group interacts strongly with watermolecules via dipole or ion-dipole interactions. Based on the nature ofthe hydrophilic group, surfactants are classified into anionic,cationic, zwitterionic, nonionic and polymeric surfactants.

Suitable surfactants include, but are not limited to, ethoxylatednonylphenol comprising 9 to 10 units of ethyleneglycol, ethoxylatedundecanol comprising 8 units of ethyleneglycol, polyoxyethylene (20)sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate,polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20)sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate,sorbitan monostearate, sorbitan monooleate, ethoxylated hydrogenatedricin oils, sodium laurylsulfate, a diblock copolymer of ethyleneoxydeand propyleneoxyde, Ethylene Oxide-Propylene Oxide Block Copolymers, andtetra-functional block copolymers based on ethylene oxide and propyleneoxide, Glyceryl monoesters, Glyceryl caprate, Glyceryl caprylate,Glyceryl cocate, Glyceryl erucate, Glyceryl hydroxysterate, Glycerylisostearate, Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate,Glyceryl myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate,Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thiglycolate, Glyceryldilaurate, Glyceryl dioleate, Glyceryl dimyristate, Glyceryl disterate,Glyceryl sesuioleate, Glyceryl stearate lactate, Polyoxyethylenecetyl/stearyl ether, Polyoxyethylene cholesterol ether, Polyoxyethylenelaurate or dilaurate, Polyoxyethylene stearate or distearate,polyoxyethylene fatty ethers, Polyoxyethylene lauryl ether,Polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, asteroid, Cholesterol, Betasitosterol, Bisabolol, fatty acid esters ofalcohols, isopropyl myristate, Aliphati-isopropyl n-butyrate, Isopropyln-hexanoate, Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecylmyristate, alkoxylated alcohols, alkoxylated acids, alkoxylated amides,alkoxylated sugar derivatives, alkoxylated derivatives of natural oilsand waxes, polyoxyethylene polyoxypropylene block copolymers,nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20 methylglucosesesquistearate, PEG40 lanolin, PEG-40 castor oil, PEG-40 hydrogenatedcastor oil, polyoxyethylene fatty ethers, glyceryl diesters,polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, andpolyoxyethylene lauryl ether, glyceryl dilaurate, glyceryl dimystate,glyceryl distearate, semi-synthetic derivatives thereof, or mixturesthereof.

Additional suitable surfactants include, but are not limited to,non-ionic lipids, such as glyceryl laurate, glyceryl myristate, glyceryldilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, andmixtures thereof.

In additional embodiments, the surfactant is a polyoxyethylene fattyether having a polyoxyethylene head group ranging from about 2 to about100 groups, or an alkoxylated alcohol having the structure R₅—(OCH₂CH₂)_(y)—OH, wherein R₅ is a branched or unbranched alkyl group havingfrom about 6 to about 22 carbon atoms and y is between about 4 and about100, and preferably, between about 10 and about 100. Preferably, thealkoxylated alcohol is the species wherein R₅ is a lauryl group and yhas an average value of 23.

In a different embodiment, the surfactant is an alkoxylated alcoholwhich is an ethoxylated derivative of lanolin alcohol. Preferably, theethoxylated derivative of lanolin alcohol is laneth-10, which is thepolyethylene glycol ether of lanolin alcohol with an averageethoxylation value of 10.

Nonionic surfactants include, but are not limited to, an ethoxylatedsurfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fattyacid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan esterethoxylated, a fatty amino ethoxylated, an ethylene oxide-propyleneoxide copolymer, Bis(polyethylene glycol bis[imidazoyl carbonyl]),nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij® 35,Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor®EL, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine,n-Decyl alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecylbeta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycolmonodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630, Igepal CA-630,Methyl-6-O-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethyleneglycol monododecyl ether, N—N-Nonanoyl-N-methylglucamine, Octaethyleneglycol monodecyl ether, Octaethylene glycol monododecyl ether,Octaethylene glycol monohexadecyl ether, Octaethylene glycolmonooctadecyl ether, Octaethylene glycol monotetradecyl ether,Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether,Pentaethylene glycol monododecyl ether, Pentaethylene glycolmonohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethyleneglycol monooctadecyl ether, Pentaethylene glycol monooctyl ether,Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-1,Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl),Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillajabark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85,Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5,Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10,Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7,Tergitol, Type NP-9, Tergitol, Tergitol, Type TMN-10, Tergitol, TypeTMN-6, Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecylether, Tetraethylene glycol monododecyl ether, Tetraethylene glycolmonotetradecyl ether, Triethylene glycol monodecyl ether, Triethyleneglycol monododecyl ether, Triethylene glycol monohexadecyl ether,Triethylene glycol monooctyl ether, Triethylene glycol monotetradecylether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, TritonGR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, TritonX-15, Triton X-151, Triton X-200, Triton X-207, Triton® X-114, Triton®X-165, Triton® X-305, Triton® X-405, Triton® X-45, Triton® X-705-70,TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61, TWEEN® 65, TWEEN®80, TWEEN® 81, TWEEN® 85, Tyloxapol, n-Undecyl beta-D-glucopyranoside,semi-synthetic derivatives thereof, or combinations thereof.

In addition, the nonionic surfactant can be a poloxamer. Poloxamers arepolymers made of a block of polyoxyethylene, followed by a block ofpolyoxypropylene, followed by a block of polyoxyethylene. The averagenumber of units of polyoxyethylene and polyoxypropylene varies based onthe number associated with the polymer. For example, the smallestpolymer, Poloxamer 101, consists of a block with an average of 2 unitsof polyoxyethylene, a block with an average of 16 units ofpolyoxypropylene, followed by a block with an average of 2 units ofpolyoxyethylene. Poloxamers range from colorless liquids and pastes towhite solids. In cosmetics and personal care products, Poloxamers areused in the formulation of skin cleansers, bath products, shampoos, hairconditioners, mouthwashes, eye makeup remover and other skin and hairproducts. Examples of Poloxamers include, but are not limited to,Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer123, Poloxamer 124, Poloxamer 181, Poloxamer 182, Poloxamer 183,Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235,Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335,Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer407, Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.

Suitable cationic surfactants include, but are not limited to, aquarternary ammonium compound, an alkyl trimethyl ammonium chloridecompound, a dialkyl dimethyl ammonium chloride compound, a cationichalogen-containing compound, such as cetylpyridinium chloride,Benzalkonium chloride, Benzalkonium chloride,Benzyldimethylhexadecylammonium chloride,Benzyldimethyltetradecylammonium chloride, Benzyldodecyldimethylammoniumbromide, Benzyltrimethylammonium tetrachloroiodate,Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammoniumbromide, Dodecyltrimethylammonium bromide, Dodecyltrimethylammoniumbromide, Ethylhexadecyldimethylammonium bromide, Girard's reagent T,Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium bromide,N,N′,N′-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzoniumbromide, Trimethyl(tetradecyl)ammonium bromide,1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, 1-Decanaminium, N-decyl-N,N-dimethyl-, chloride, Didecyl dimethyl ammonium chloride,2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride, Alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride, Alkyl bis(2-hydroxyethyl) benzyl ammonium chloride, Alkyldemethyl benzyl ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% C12), Alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% C14, 40% C12, 10% C16), Alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C14, 23% C12, 20% C16), Alkyl dimethyl benzylammonium chloride, Alkyl dimethyl benzyl ammonium chloride (100% C14),Alkyl dimethyl benzyl ammonium chloride (100% C16), Alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12), Alkyl dimethyl benzylammonium chloride (47% C12, 18% C14), Alkyl dimethyl benzyl ammoniumchloride (55% C16, 20% C14), Alkyl dimethyl benzyl ammonium chloride(58% C14, 28% C16), Alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12), Alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14),Alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14), Alkyldimethyl benzyl ammonium chloride (65% C12, 25% C14), Alkyl dimethylbenzyl ammonium chloride (67% C12, 24% C14), Alkyl dimethyl benzylammonium chloride (67% C12, 25% C14), Alkyl dimethyl benzyl ammoniumchloride (90% C14, 5% C12), Alkyl dimethyl benzyl ammonium chloride (93%C14, 4% C12), Alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18),Alkyl didecyl dimethyl ammonium chloride, Alkyl dimethyl benzyl ammoniumchloride (C12-16), Alkyl dimethyl benzyl ammonium chloride (C12-18),dialkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl dimethybenzylammonium chloride, Alkyl dimethyl ethyl ammonium bromide (90% C14, 5%C16, 5% C12), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl andalkenyl groups as in the fatty acids of soybean oil), Alkyl dimethylethylbenzyl ammonium chloride, Alkyl dimethyl ethylbenzyl ammoniumchloride (60% C14), Alkyl dimethyl isopropylbenzyl ammonium chloride(50% C12, 30% C14, 17% C16, 3% C18), Alkyl trimethyl ammonium chloride(58% C18, 40% C16, 1% C14, 1% C12), Alkyl trimethyl ammonium chloride(90% C18, 10% C16), Alkyldimethyl(ethylbenzyl) ammonium chloride(C12-18), Di-(C8-10)-alkyl dimethyl ammonium chlorides, Dialkyl dimethylammonium chloride, Dialkyl methyl benzyl ammonium chloride, Didecyldimethyl ammonium chloride, Diisodecyl dimethyl ammonium chloride,Dioctyl dimethyl ammonium chloride, Dodecyl bis (2-hydroxyethyl) octylhydrogen ammonium chloride, Dodecyl dimethyl benzyl ammonium chloride,Dodecylcarbamoyl methyl dinethyl benzyl ammonium chloride, Heptadecylhydroxyethylimidazolinium chloride,Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium chloride(and) Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammonium chloridepolymer, n-Tetradecyl dimethyl benzyl ammonium chloride monohydrate,Octyl decyl dimethyl ammonium chloride, Octyl dodecyl dimethyl ammoniumchloride, Octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride,Oxydiethylenebis(alkyl dimethyl ammonium chloride), Trimethoxysilypropyl dimethyl octadecyl ammonium chloride, Trimethoxysilyl quats,Trimethyl dodecylbenzyl ammonium chloride, semi-synthetic derivativesthereof, and combinations thereof.

Exemplary cationic halogen-containing compounds include, but are notlimited to, cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides, ortetradecyltrimethylammonium halides. In some particular embodiments,suitable cationic halogen containing compounds comprise, but are notlimited to, cetylpyridinium chloride (CPC), cetyltrimethylammoniumchloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide(CPB), cetyltrimethylammonium bromide (CTAB), cetyidimethylethylammoniumbromide, cetyltributylphosphonium bromide, dodecyltrimethylammoniumbromide, and tetrad ecyltrimethylammonium bromide. In particularlypreferred embodiments, the cationic halogen containing compound is CPC,although the compositions of the present invention are not limited toformulation with a particular cationic containing compound.

Suitable anionic surfactants include, but are not limited to, acarboxylate, a sulphate, a sulphonate, a phosphate, chenodeoxycholicacid, chenodeoxycholic acid sodium salt, cholic acid, ox or sheep bile,Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid, Deoxycholic acidmethyl ester, Digitonin, Digitoxigenin, N,N-DimethyldodecylamineN-oxide, Docusate sodium salt, Glycochenodeoxycholic acid sodium salt,Glycocholic acid hydrate, synthetic, Glycocholic acid sodium salthydrate, synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholicacid sodium salt, Glycolithocholic acid 3-sulfate disodium salt,Glycolithocholic acid ethyl ester, N-Lauroylsarcosine sodium salt,N-Lauroylsarcosine solution, N-Lauroylsarcosine solution, Lithiumdodecyl sulfate, Lithium dodecyl sulfate, Lithium dodecyl sulfate, Lugolsolution, Niaproof 4, Type 4, 1-Octanesulfonic acid sodium salt, Sodium1-butanesulfonate, Sodium 1-decanesulfonate, Sodium 1-decanesulfonate,Sodium 1-dodecanesulfonate, Sodium 1-heptanesulfonate anhydrous, Sodium1-heptanesulfonate anhydrous, Sodium 1-nonanesulfonate, Sodium1-propanesulfonate monohydrate, Sodium 2-bromoethanesulfonate, Sodiumcholate hydrate, Sodium choleate, Sodium deoxycholate, Sodiumdeoxycholate monohydrate, Sodium dodecyl sulfate, Sodium hexanesulfonateanhydrous, Sodium octyl sulfate, Sodium pentanesulfonate anhydrous,Sodium taurocholate, Taurochenodeoxycholic acid sodium salt,Taurodeoxycholic acid sodium salt monohydrate, Taurohyodeoxycholic acidsodium salt hydrate, Taurolithocholic acid 3-sulfate disodium salt,Tauroursodeoxycholic acid sodium salt, Trizma® dodecyl sulfate, TWEEN®80, Ursodeoxycholic acid, semi-synthetic derivatives thereof, andcombinations thereof.

Suitable zwitterionic surfactants include, but are not limited to, anN-alkyl betaine, lauryl amindo propyl dimethyl betaine, an alkyldimethyl glycinate, an N-alkyl amino propionate, CHAPS, minimum 98%(TLC), CHAPS, SigmaUltra, minimum 98% (TLC), CHAPS, for electrophoresis,minimum 98% (TLC), CHAPSO, minimum 98%, CHAPSO, SigmaUltra, CHAPSO, forelectrophoresis, 3-(Decyldimethylammonio)propanesulfonate inner salt,3-Dodecyldimethylammonio)propanesulfonate inner salt, SigmaUltra,3-(Dodecyldimethylammonio)propanesulfonate inner salt,3-(N,N-Dimethylmyristylammonio)propanesulfonate,3-(N,N-Dimethyloctadecylammonio)propanesulfonate,3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,3-(N,N-Dimethylpalmitylammonio)propanesulfonate, semi-syntheticderivatives thereof, and combinations thereof.

In some embodiments, the nanoemulsion comprises a cationic surfactant,which can be cetylpyridinium chloride. In other embodiments of theinvention, the nanoemulsion comprises a cationic surfactant, and theconcentration of the cationic surfactant is less than about 5.0% andgreater than about 0.001%. In yet another embodiment of the invention,the nanoemulsion comprises a cationic surfactant, and the concentrationof the cationic surfactant is selected from the group consisting of lessthan about 5%, less than about 4.5%, less than about 4.0%, less thanabout 3.5%, less than about 3.0%, less than about 2.5%, less than about2.0%, less than about 1.5%, less than about 1.0%, less than about 0.90%,less than about 0.80%, less than about 0.70%, less than about 0.60%,less than about 0.50%, less than about 0.40%, less than about 0.30%,less than about 0.20%, or less than about 0.10%. Further, theconcentration of the cationic agent in the nanoemulsion is greater thanabout 0.002%, greater than about 0.003%, greater than about 0.004%,greater than about 0.005%, greater than about 0.006%, greater than about0.007%, greater than about 0.008%, greater than about 0.009%, greaterthan about 0.010%, or greater than about 0.001%. In one embodiment, theconcentration of the cationic agent in the nanoemulsion is less thanabout 5.0% and greater than about 0.001%.

In another embodiment of the invention, the nanoemulsion comprises atleast one cationic surfactant and at least one non-cationic surfactant.The non-cationic surfactant is a nonionic surfactant, such as apolysorbate (Tween), such as polysorbate 80 or polysorbate 20. In oneembodiment, the non-ionic surfactant is present in a concentration ofabout 0.05% to about 7.0%, or the non-ionic surfactant is present in aconcentration of about 0.5% to about 4%. In yet another embodiment ofthe invention, the nanoemulsion comprises a cationic surfactant presentin a concentration of about 0.01% to about 2%, in combination with anonionic surfactant.

5. Additional Ingredients

Additional compounds suitable for use in the nanoemulsions of theinvention include but are not limited to one or more solvents, such asan organic phosphate-based solvent, bulking agents, coloring agents,pharmaceutically acceptable excipients, a preservative, pH adjuster,buffer, chelating agent, etc. The additional compounds can be admixedinto a previously emulsified nanoemulsion, or the additional compoundscan be added to the original mixture to be emulsified. In certain ofthese embodiments, one or more additional compounds are admixed into anexisting nanoemulsion composition immediately prior to its use.

Suitable preservatives in the nanoemulsions of the invention include,but are not limited to, cetylpyridinium chloride, benzalkonium chloride,benzyl alcohol, chlorhexidine, imidazolidinyl urea, phenol, potassiumsorbate, benzoic acid, bronopol, chlorocresol, paraben esters,phenoxyethanol, sorbic acid, alpha-tocophernol, ascorbic acid, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, sodiumascorbate, sodium metabisulphite, citric acid, edetic acid,semi-synthetic derivatives thereof, and combinations thereof. Othersuitable preservatives include, but are not limited to, benzyl alcohol,chlorhexidine (bis (p-chlorophenyldiguanido) hexane), chlorphenesin(3-(-4-chloropheoxy)-propane-1,2-diol), Kathon CG (methyl andmethylchloroisothiazolinone), parabens (methyl, ethyl, propyl, butylhydrobenzoates), phenoxyethanol (2-phenoxyethanol), sorbic acid(potassium sorbate, sorbic acid), Phenonip (phenoxyethanol, methyl,ethyl, butyl, propyl parabens), Phenoroc (phenoxyethanol 0.73%, methylparaben 0.2%, propyl paraben 0.07%), Liquipar Oil (isopropyl, isobutyl,butylparabens), Liquipar PE (70% phenoxyethanol, 30% liquipar oil),Nipaguard MPA (benzyl alcohol (70%), methyl & propyl parabens),Nipaguard MPS (propylene glycol, methyl & propyl parabens), Nipasept(methyl, ethyl and propyl parabens), Nipastat (methyl, butyl, ethyl andpropyel parabens), Elestab 388 (phenoxyethanol in propylene glycol pluschlorphenesin and methylparaben), and Killitol (7.5% chlorphenesin and7.5% methyl parabens).

The nanoemulsion may further comprise at least one pH adjuster. SuitablepH adjusters in the nanoemulsion of the invention include, but are notlimited to, diethyanolamine, lactic acid, monoethanolamine,triethylanolamine, sodium hydroxide, sodium phosphate, semi-syntheticderivatives thereof, and combinations thereof.

In addition, the nanoemulsion can comprise a chelating agent. In oneembodiment of the invention, the chelating agent is present in an amountof about 0.0005% to about 1.0%. Examples of chelating agents include,but are not limited to, phytic acid, polyphosphoric acid, citric acid,gluconic acid, acetic acid, lactic acid, ethylenediamine,ethylenediaminetetraacetic acid (EDTA), and dimercaprol, and a preferredchelating agent is ethylenediaminetetraacetic acid.

The nanoemulsion can comprise a buffering agent, such as apharmaceutically acceptable buffering agent. Examples of bufferingagents include, but are not limited to,2-Amino-2-methyl-1,3-propanediol, ≧99.5% (NT),2-Amino-2-methyl-1-propanol, ≧99.0% (GC), L-(+)-Tartaric acid, ≧99.5%(T), ACES, ≧99.5% (T), ADA, ≧99.0% (T), Acetic acid, ≧99.5% (GC/T),Acetic acid, for luminescence, ≧99.5% (GC/T), Ammonium acetate solution,for molecular biology, −5 M in H₂O, Ammonium acetate, for luminescence,≧99.0% (calc. on dry substance, T), Ammonium bicarbonate, ≧99.5% (T),Ammonium citrate dibasic, ≧99.0% (T), Ammonium formate solution, 10 M inH₂O, Ammonium formate, ≧99.0% (calc. based on dry substance, NT),Ammonium oxalate monohydrate, ≧99.5% (RT), Ammonium phosphate dibasicsolution, 2.5 M in H₂O, Ammonium phosphate dibasic, ≧99.0% (T), Ammoniumphosphate monobasic solution, 2.5 M in H₂O, Ammonium phosphatemonobasic, ≧99.5% (T), Ammonium sodium phosphate dibasic tetrahydrate,≧99.5% (NT), Ammonium sulfate solution, for molecular biology, 3.2 M inH₂O, Ammonium tartrate dibasic solution, 2 M in H₂O (colorless solutionat 20° C.), Ammonium tartrate dibasic, ≧99.5% (T), BES buffered saline,for molecular biology, 2× concentrate, BES, ≧99.5% (T), BES, formolecular biology, ≧99.5% (T), BICINE buffer Solution, for molecularbiology, 1 M in H₂O, BICINE, ≧99.5% (T), BIS-TRIS, ≧99.0% (NT),Bicarbonate buffer solution, ≧0.1 M Na₂CO₃, ≧0.2 M NaHCO₃, Boric acid,≧99.5% (T), Boric acid, for molecular biology, ≧99.5% (T), CAPS, ≧99.0%(TLC), CHES, ≧99.5% (T), Calcium acetate hydrate, ≧99.0% (calc. on driedmaterial, KT), Calcium carbonate, precipitated, ≧99.0% (KT), Calciumcitrate tribasic tetrahydrate, ≧98.0% (calc. on dry substance, KT),Citrate Concentrated Solution, for molecular biology, 1 M in H₂O, Citricacid, anhydrous, ≧99.5% (T), Citric acid, for luminescence, anhydrous,≧99.5% (T), Diethanolamine, ≧99.5% (GC), EPPS, ≧99.0% (T),

Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecularbiology, ≧99.0% (T), Formic acid solution, 1.0 M in H₂O, Gly-Gly-Gly,≧99.0% (NT), Gly-Gly, ≧99.5% (NT), Glycine, ≧99.0% (NT), Glycine, forluminescence, ≧99.0% (NT), Glycine, for molecular biology, ≧99.0% (NT),HEPES buffered saline, for molecular biology, 2× concentrate, HEPES,≧99.5% (T), HEPES, for molecular biology, ≧99.5% (T), Imidazole bufferSolution, 1 M in H₂O, Imidazole, ≧99.5% (GC), Imidazole, forluminescence, ≧99.5% (GC), Imidazole, for molecular biology, ≧99.5%(GC), Lipoprotein Refolding Buffer, Lithium acetate dihydrate, ≧99.0%(NT), Lithium citrate tribasic tetrahydrate, ≧99.5% (NT), MES hydrate,≧99.5% (T), MES monohydrate, for luminescence, ≧99.5% (T), MES solution,for molecular biology, 0.5 M in H₂O, MOPS, ≧99.5% (T), MOPS, forluminescence, ≧99.5% (T), MOPS, for molecular biology, ≧99.5% (T),Magnesium acetate solution, for molecular biology, ˜1 M in H₂O,Magnesium acetate tetrahydrate, ≧99.0% (KT), Magnesium citrate tribasicnonahydrate, ≧98.0% (calc. based on dry substance, KT), Magnesiumformate solution, 0.5 M in H₂O, Magnesium phosphate dibasic trihydrate,≧98.0% (KT), Neutralization solution for the in-situ hybridization forin-situ hybridization, for molecular biology, Oxalic acid dihydrate,≧99.5% (RT), PIPES, ≧99.5% (T), PIPES, for molecular biology, ≧99.5%(T), Phosphate buffered saline, solution (autoclaved), Phosphatebuffered saline, washing buffer for peroxidase conjugates in WesternBlotting, 10× concentrate, Piperazine, anhydrous, ≧99.0% (T), PotassiumD-tartrate monobasic, ≧99.0% (T), Potassium acetate solution, formolecular biology, Potassium acetate solution, for molecular biology, 5M in H₂O, Potassium acetate solution, for molecular biology, ˜1 M inH₂O, Potassium acetate, ≧99.0% (NT), Potassium acetate, forluminescence, ≧99.0% (NT), Potassium acetate, for molecular biology,≧99.0% (NT), Potassium bicarbonate, ≧99.5% (T), Potassium carbonate,anhydrous, ≧99.0% (T), Potassium chloride, ≧99.5% (AT), Potassiumcitrate monobasic, ≧99.0% (dried material, NT), Potassium citratetribasic solution, 1 M in H₂O, Potassium formate solution, 14 M in H₂O,Potassium formate, ≧99.5% (NT), Potassium oxalate monohydrate, ≧99.0%(RT), Potassium phosphate dibasic, anhydrous, ≧99.0% (T), Potassiumphosphate dibasic, for luminescence, anhydrous, ≧99.0% (T), Potassiumphosphate dibasic, for molecular biology, anhydrous, ≧99.0% (T),Potassium phosphate monobasic, anhydrous, ≧99.5% (T), Potassiumphosphate monobasic, for molecular biology, anhydrous, ≧99.5% (T),Potassium phosphate tribasic monohydrate, ≧95% (T), Potassium phthalatemonobasic, ≧99.5% (T), Potassium sodium tartrate solution, 1.5 M in H₂O,Potassium sodium tartrate tetrahydrate, ≧99.5% (NT), Potassiumtetraborate tetrahydrate, ≧99.0% (T), Potassium tetraoxalate dihydrate,≧99.5% (RT), Propionic acid solution, 1.0 M in H₂O, STE buffer solution,for molecular biology, pH 7.8, STET buffer solution, for molecularbiology, pH 8.0, Sodium 5,5-diethylbarbiturate, ≧99.5% (NT), Sodiumacetate solution, for molecular biology, ˜3 M in H₂O, Sodium acetatetrihydrate, ≧99.5% (NT), Sodium acetate, anhydrous, ≧99.0% (NT), Sodiumacetate, for luminescence, anhydrous, ≧99.0% (NT), Sodium acetate, formolecular biology, anhydrous, ≧99.0% (NT), Sodium bicarbonate, ≧99.5%(T), Sodium bitartrate monohydrate, ≧99.0% (T), Sodium carbonatedecahydrate, ≧99.5% (T), Sodium carbonate, anhydrous, ≧99.5% (calc. ondry substance, T), Sodium citrate monobasic, anhydrous, ≧99.5% (T),Sodium citrate tribasic dihydrate, ≧99.0% (NT), Sodium citrate tribasicdihydrate, for luminescence, ≧99.0% (NT), Sodium citrate tribasicdihydrate, for molecular biology, ≧99.5% (NT), Sodium formate solution,8 M in H₂O, Sodium oxalate, ≧99.5% (RT), Sodium phosphate dibasicdihydrate, ≧99.0% (T), Sodium phosphate dibasic dihydrate, forluminescence, ≧99.0% (T), Sodium phosphate dibasic dihydrate, formolecular biology, ≧99.0% (T), Sodium phosphate dibasic dodecahydrate,≧99.0% (T), Sodium phosphate dibasic solution, 0.5 M in H₂O, Sodiumphosphate dibasic, anhydrous, ≧99.5% (T), Sodium phosphate dibasic, formolecular biology, ≧99.5% (T), Sodium phosphate monobasic dihydrate,≧99.0% (T), Sodium phosphate monobasic dihydrate, for molecular biology,≧99.0% (T), Sodium phosphate monobasic monohydrate, for molecularbiology, ≧99.5% (T), Sodium phosphate monobasic solution, 5 M in H₂O,Sodium pyrophosphate dibasic, ≧99.0% (T), Sodium pyrophosphatetetrabasic decahydrate, ≧99.5% (T), Sodium tartrate dibasic dihydrate,≧99.0% (NT), Sodium tartrate dibasic solution, 1.5 M in H₂O (colorlesssolution at 20° C.), Sodium tetraborate decahydrate, ≧99.5% (T), TAPS,≧99.5% (T), TES, ≧99.5% (calc. based on dry substance, T), TM buffersolution, for molecular biology, pH 7.4, TNT buffer solution, formolecular biology, pH 8.0, TRIS Glycine buffer solution, 10×concentrate, TRIS acetate—EDTA buffer solution, for molecular biology,TRIS buffered saline, 10× concentrate, TRIS glycine SDS buffer solution,for electrophoresis, 10× concentrate, TRIS phosphate—EDTA buffersolution, for molecular biology, concentrate, 1 Ox concentrate, Tricine,≧99.5% (NT), Triethanolamine, ≧99.5% (GC), Triethylamine, ≧99.5% (GC),Triethylammonium acetate buffer, volatile buffer, ˜1.0 M in H₂O,Triethylammonium phosphate solution, volatile buffer, ˜1.0 M in H₂O,Trimethylammonium acetate solution, volatile buffer, ˜1.0 M in H₂O,Trimethylammonium phosphate solution, volatile buffer, ˜1 M in H₂O,Tris-EDTA buffer solution, for molecular biology, concentrate, 100×concentrate, Tris-EDTA buffer solution, for molecular biology, pH 7.4,Tris-EDTA buffer solution, for molecular biology, pH 8.0, Trizma®acetate, ≧99.0% (NT), Trizma® base, ≧99.8% (T), Trizma® base, ≧99.8%(T), Trizma® base, for luminescence, ≧99.8% (T), Trizma® base, formolecular biology, ≧99.8% (T), Trizma® carbonate, ≧98.5% (T), Trizma®hydrochloride buffer solution, for molecular biology, pH 7.2, Trizma®hydrochloride buffer solution, for molecular biology, pH 7.4, Trizma®hydrochloride buffer solution, for molecular biology, pH 7.6, Trizma®hydrochloride buffer solution, for molecular biology, pH 8.0, Trizma®hydrochloride, ≧99.0% (AT), Trizma® hydrochloride, for luminescence,≧99.0% (AT), Trizma® hydrochloride, for molecular biology, ≧99.0% (AT),and Trizma® maleate, ≧99.5% (NT).

The nanoemulsion can comprise one or more emulsifying agents to aid inthe formation of emulsions. Emulsifying agents include compounds thataggregate at the oil/water interface to form a kind of continuousmembrane that prevents direct contact between two adjacent droplets.Certain embodiments of the present invention feature nanoemulsions thatmay readily be diluted with water to a desired concentration withoutimpairing their antiviral properties.

6. Active Agents Incorporated into a Nanoemulsion of the Invention

In a further embodiment of the invention, a nanoemulsion comprises anadditional active agent, such as an antibiotic or a palliative agent(such as for burn wound treatment). Addition of another agent mayenhance the therapeutic effectiveness of the nanoemulsion. Thenanoemulsion in and of itself has anti-bacterial activity and does notneed to be combined with another active agent to obtain therapeuticeffectiveness. Any antibacterial (or antibiotic) agent suitable fortreating a bacterial infection can be incorporated into the topicalnanoemulsions of the invention.

Examples of such antibiotic agents include, but are not limited to,aminoglycosides, Ansamycins, Carbacephems, Carbapenems, Cephalosporins,Glycopeptides, Macrolides, Monobactams, Penicillins, Polypeptides,Polymyxin, Quinolones, Sulfonamides, Tetracyclines, and others (e.g.,Arsphenamine, Chloramphenicol, Clindamycin, Lincomycin, Ethambutol,Fosfomycin, Fusidic acid, Furazolidone, Isoniazid, Linezolid,Metronidazole, Mupirocin, Nitrofurantoin, Platensimycin, Pyrazinamide,Quinupristin/Dalfopristin, Rifampicin (Rifampin in US), Thiamphenicol,Tinidazole, Dapsone, and lofazimine).

Examples of these classes of antibiotics include, but are not limitedto, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin,Tobramycin, Paromomycin, Geldanamycin, Herbimycin, Loracarbef,Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil,Cefazolin, Cefalotin or Cefalothin, Cefalexin, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,Ceftizoxime, Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin,Vancomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin,Roxithromycin, Troleandomycin, Telithromycin, Spectinomycin, Aztreonam,Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin,Dicloxacillin, Flucloxacillin, Mezlocillin, Meticillin, Nafcillin,Oxacillin, Penicillin, Piperacillin, Ticarcillin, Bacitracin, Colistin,Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin, Trovafloxacin,Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide,Sulfonamidochrysoidine (archaic), Sulfacetamide, Sulfadiazine,Sulfamethizole, Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole,Trimethoprim, rimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX),Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, andTetracycline.

Examples of palliative agents which may be incorporated into thenanoemulsions of the invention include, but are not limited to, menthol,camphor, phenol, allantoin, benzocaine, corticosteroids, phenol, zincoxide, camphor, pramoxine, dimethicone, meradimate, octinoxate,octisalate, oxybenzone, dyclonine, alcohols (e.g., benzyl alcohol),mineral oil, propylene glycol, titanium dioxide, silver nitrate (AgNO₃),silver sulfadiazine, mafenide acetate, nanocrystalline impregnatedsilver dressings, a p38 MAPK inhibitor, and magnesium stearate.

D. Pharmaceutical Compositions

The nanoemulsions of the invention may be formulated into pharmaceuticalcompositions that comprise the nanoemulsion in a therapeuticallyeffective amount and suitable, pharmaceutically-acceptable excipientsfor administration to a human subject in need thereof using anyconventional pharmaceutical method of administration. Such excipientsare well known in the art.

By the phrase “therapeutically effective amount” it is meant any amountof the nanoemulsion that is effective in treating microorganisms bykilling or inhibiting the growth of the microorganisms, causing themicroorganisms to lose pathogenicity, or any combination thereof.

Exemplary dosage forms may include, but are not limited to, patches,ointments, creams, emulsions, liquids, lotions, gels, bioadhesive gels,aerosols, pastes, foams, sunscreens, capsules, microcapsules, or in theform of an article or carrier, such as a bandage, insert, syringe-likeapplicator, pessary, powder, and talc or other solid.

The pharmaceutical compositions may be formulated for immediate release,sustained release, controlled release, delayed release, or anycombinations thereof. In some embodiments, the formulations may comprisea penetration-enhancing agent for enhancing penetration of thenanoemulsion through the stratum corneum and into the epidermis ordermis (i.e., for methods of treating burn wounds). Suitablepenetration-enhancing agents include, but are not limited to, alcoholssuch as ethanol, triglycerides and aloe compositions. The amount of thepenetration-enhancing agent may comprise from about 0.5% to about 40% byweight of the formulation.

When appropriate, for example when treating burn wounds, thenanoemulsions of the invention can be applied and/or delivered utilizingelectrophoretic delivery/electrophoresis. Such transdermal methods,which comprise applying an electrical current, are well known in theart.

In another embodiment of the invention, minimal systemic absorption ofthe nanoemulsion occurs upon topical administration. Such minimalsystemic exposure can be determined by the detection of less than 10ng/mL, less than 8 ng/mL, less than 5 ng/mL, less than 4 ng/mL, lessthan 3 ng/mL, or less than 2 ng/mL of the one or more surfactantspresent in the nanoemulsion in the plasma of the subject. Lack ofsystemic absorption may be monitored, for example, by measuring theamount of the surfactant, such as the cationic surfactant, in the plasmaof the human subject undergoing treatment. Amounts of surfactant ofequal to or less than about 10 ng/ml in the plasma confirms minimalsystemic absorption.

The pharmaceutical compositions may be applied in a singleadministration or in multiple administrations. The pharmaceuticalcompositions can be applied for at least one day, at least two days atleast three days at least four days at least 5 days, once a week, atleast twice a week, at least once a day, at least twice a day, multipletimes daily, multiple times weekly, biweekly, at least once a month, orany combination thereof.

Following topical or intradermal administration, the nanoemulsion may beoccluded or semi-occluded. Occlusion or semi-occlusion may be performedby overlaying a bandage, polyoleofin film, article of clothing,impermeabile barrier, or semi-impermeable barrier to the topicalpreparation.

Exemplary Nano Emulsions

Several exemplary nanoemulsions are described below, although themethods of the invention are not limited to the use of suchnanoemulsions. The components and quantity of each can be varied asdescribed herein in the preparation of other nanoemulsions. (“CPC”refers to cetylpyridinium chloride, which is a cationic surfactantpresent in the nanoemulsions). Compositions are w/w % unless otherwisenoted.

TABLE 1 Exemplary Nanoemulsions Nanoemulsion Component Weight PercentW₂₀5EC ED Distilled Water 23.418% EDTA 0.0745% Cetylpyridinium Chloride 1.068% Tween 20  5.92% Ethanol  6.73% Soybean Oil  62.79% P₄₀₇5ECDistilled Water  23.49% CPC  1.068% Poloxamer 407  5.92% Ethanol  6.73%Soybean Oil, NP  62.79% W₂₀5GBA₂ (v/v%) Distilled Water  20.93% BTC 824   2% Tween 20    5% Glycerine    8% Soybean Oil    64% W₈₀5EC Water23.490% Ethanol  6.730% Cetylpyridinium Chloride  1.068% Polysorbate 80 5.920% Refined Soybean Oil 62.790% W₂₀5ECEDL2 Distilled Water 23.418%EDTA 0.0745% Cetylpyridinium Chloride  1.068% Tween 20  5.92% Ethanol 6.73% Soybean Oil  62.79% W₂₀5GBA₂ED (v/v%) Distilled Water  20.93%EDTA 0.0745% BTC 824    2% Tween 20    5% Glycerine    8% Soybean Oil   64%

The following nanoemulsions have an average particle (droplet) size ofabout 300 nm to about 600 nm: W₂₀5EC ED, P₄₀₇5EC, W₂₀5GBA₂, W₈₀5EC, andW₂₀5GBA₂ED. The W₂₀5ECEDL2, which undergoes high pressure processing,has an average particle (droplet) size of about 150 nm to about 400 nm.The formulations listed in the table above are “neat” or “concentrated”formulations, meaning that the formulation intended for therapeutic usecan be diluted as desired.

Methods of Manufacture

The nanoemulsions of the invention can be formed using classic emulsionforming techniques. See e.g., U.S. 2004/0043041. See also U.S. Pat. Nos.6,015,832, 6,506,803, 6,559,189, 6,635,676, and US Patent PublicationNo. 20040043041, all of which are incorporated by reference. Inaddition, methods of making emulsions are described in U.S. Pat. Nos.5,103,497 and 4,895,452 (herein incorporated by reference). In anexemplary method, the oil is mixed with the aqueous phase underrelatively high shear forces (e.g., using high hydraulic and mechanicalforces) to obtain a nanoemulsion comprising oil droplets having anaverage diameter of less than about 1000 nm. Some embodiments of theinvention employ a nanoemulsion having an oil phase comprising analcohol such as ethanol. The oil and aqueous phases can be blended usingany apparatus capable of producing shear forces sufficient to form anemulsion, such as French Presses or high shear mixers (e.g., FDAapproved high shear mixers are available, for example, from Admix, Inc.,Manchester, N.H.). Methods of producing such emulsions are described inU.S. Pat. Nos. 5,103,497 and 4,895,452, herein incorporated by referencein their entireties.

In an exemplary embodiment, the nanoemulsions used in the methods of theinvention comprise droplets of an oily discontinuous phase dispersed inan aqueous continuous phase, such as water. The nanoemulsions of theinvention are stable, and do not decompose even after long storageperiods. Certain nanoemulsions of the invention are non-toxic and safewhen swallowed, inhaled, or contacted to the skin of a subject.

The compositions of the invention can be produced in large quantitiesand are stable for many months at a broad range of temperatures. Thenanoemulsion can have textures ranging from that of a semi-solid creamto that of a thin lotion, and can be applied topically by hand, and canbe sprayed onto a surface or nebulized.

As stated above, at least a portion of the emulsion may be in the formof lipid structures including, but not limited to, unilamellar,multilamellar, and paucliamellar lipid vesicles, micelles, and lamellarphases.

The present invention contemplates that many variations of the describednanoemulsions will be useful in the methods of the present invention. Todetermine if a candidate nanoemulsion is suitable for use with thepresent invention, three criteria are analyzed. Using the methods andstandards described herein, candidate emulsions can be easily tested todetermine if they are suitable. First, the desired ingredients areprepared using the methods described herein, to determine if ananoemulsion can be formed. If a nanoemulsion cannot be formed, thecandidate is rejected. Second, the candidate nanoemulsion should form astable emulsion. A nanoemulsion is stable if it remains in emulsion formfor a sufficient period to allow its intended use. For example, fornanoemulsions that are to be stored, shipped, etc., it may be desiredthat the nanoemulsion remain in emulsion form for months to years.Typical nanoemulsions that are relatively unstable, will lose their formwithin a day. Third, the candidate nanoemulsion should have efficacy forits intended use. For example, the emulsions of the invention shouldkill or disable microorganisms in vitro. To determine the suitability ofa particular candidate nanoemulsion against a desired microorganism, thenanoemulsion is exposed to the microorganism for one or more timeperiods in a side-by-side experiment with an appropriate control sample(e.g., a negative control such as water) and determining if, and to whatdegree, the nanoemulsion kills or disables the microorganism.

The nanoemulsion of the invention can be provided in many differenttypes of containers and delivery systems. For example, in someembodiments of the invention, the nanoemulsions are provided as aliquid, lotion, cream or other solid or semi-solid form. Thenanoemulsions of the invention may be incorporated into hydrogelformulations.

The nanoemulsions can be delivered (e.g., to a subject or customers) inany suitable container. Suitable containers can be used that provide oneor more single use or multi-use dosages of the nanoemulsion for thedesired application. In some embodiments of the invention, thenanoemulsions are provided in a suspension or liquid form. Suchnanoemulsions can be delivered in any suitable container including spraybottles (e.g., pressurized spray bottles, nebulizers).

Exemplary Methods of Use

As described in more detail throughout this application, the presentinvention is directed to methods of treating and/or preventing arespiratory infection in a subject having Cystic fibrosis (CF). Ingeneral, the method comprises administering a nanoemulsion to thesubject, wherein the nanoemulsion comprises: (i) water; (ii) at leastone organic solvent; (iii) at least one surfactant; and (iv) at leastone oil; and wherein the nanoemulsion comprises droplets having anaverage diameter of less than about 1000 nm. In one embodiment of theinvention, the subject is susceptible to or has an infection by one ormore bacterial species selected from the group consisting ofStaphylococcus spp., Haemophilus spp., Pseudomonas spp., Burkholderiaspp., Acinetobacter spp, Stenotrophomonas spp., Escherichia spp.,Klebsiella spp., and Proteus spp. The nanoemulsion can be deliveredusing any pharmaceutically acceptable means, with inhalation being oneexample of a useful administration method.

In yet another embodiment, the invention is directed to a method oftreating or preventing an infection in a subject having a burn wound,wherein: (a) the method comprises administering a nanoemulsion to thesubject; and (b) the nanoemulsion comprises: (i) water; (ii) at leastone organic solvent; (iii) at least one surfactant; and (iv) at leastone oil; and wherein the nanoemulsion comprises droplets having anaverage diameter of less than about 1000 nm. In one embodiment of theinvention, the subject is susceptible to or has an infection by one ormore gram-negative or gram-positive bacterial species. In anotherembodiment, the bacterial species are selected from the group consistingof Staphylococcus spp., Haemophilus spp., Pseudomonas spp., Burkholderiaspp., Acinetobacter spp, Stenotrophomonas spp., Escherichia spp.,Klebsiella spp., and Proteus spp. The nanoemulsion can be deliveredusing any pharmaceutically acceptable means, with inhalation,nebulization, and topical application to mucosal surfaces being examplesof useful administration methods.

In yet another embodiment, the invention is directed to a method oftreating or preventing an Haemophilus influenzae infection in a subjectwherein: (a) the method comprises administering a nanoemulsion to thesubject having or at risk of having a Haemophilus influenzae infection;(b) the nanoemulsion comprises: (i) water; (ii) at least one organicsolvent; (iii) at least one surfactant; and (iv) at least one oil; and(c) wherein the nanoemulsion comprises droplets having an averagediameter of less than about 1000 nm. The nanoemulsion can be deliveredusing any pharmaceutically acceptable means, with inhalation,nebulization, and delivery to a mucosal surface being examples of usefuladministration methods.

In one embodiment of the invention, the nanoemulsion exhibits minimal orno toxicity or side effects. Preferably, the nanoemulsion does notexhibit resistance to bacteria. This embodiment applies to all methodsdescribed herein.

If the method relates to a respiratory infection, then in one embodimentthe respiratory infection may be associated with a bacterial biofilm,such as a biofilm present in the lungs of a subject.

All of the methods of the invention may further comprise administeringone or more antibiotics either before, during, or after administrationof the nanoemulsion. In yet another embodiment, one or more antibioticsmay be incorporated into a nanoemulsion. In yet another embodiment ofthe invention, the nanoemulsion does not exhibit any antagonism with theantibiotic.

In one embodiment of the invention, administration of a nanoemulsion andat least one antibiotic is synergistic as defined by a fractionalinhibitory concentration (FIC) index, a fractional bactericidalconcentration (FBC) index, or a combination thereof. This embodimentapplies to all methods described herein. Examples of such antibioticsinclude, but are not limited to polymyxins (colistin) andaminoglycosides (tobramycin).

In yet another embodiment, the methods of the invention may be used totreat or prevent infection by one or more bacterial species selectedfrom the group consisting of Pseudomonas aeruginosa, B. cenocepacia, A.baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, H.influenzae, E. coli, K. pneumoniae, and Proteus mirabilis. All othergram positive or gram negative bacteria are also encompassed by themethods of the invention.

In one embodiment, the minimum inhibitory concentration (MIC), theminimum bactericidal concentration (MBC), or a combination thereof forthe nanoemulsion demonstrate bacteriostatic or bactericidal activity forthe nanoemulsion. This embodiment applies to all methods describedherein.

In another embodiment of the invention, one or more bacterial speciesmay exhibit resistance against one or more antibiotics. For example, thebacterial species can be methicillin-resistant Staphylococcus aureus(MRSA). This embodiment applies to all methods described herein.

The present invention is not limited by the type of subject administereda composition of the present invention. Each of the subjects (e.g.,susceptible to respiratory infection) described above may beadministered a composition of the present invention. In addition, thecompositions and methods of the present invention are useful in thetreatment of other respiratory diseases and disorders, such as acutebronchitis, bronchiectasis, pneumonia (including ventilator-associatedpneumonia, nosocomial pneumonia, viral pneumonia, bacterial pneumonia,mycobacterial pneumonia, fungal pneumonia, eosinophilic pneumonia, andPneumocystis carinii pneumonia), tuberculosis, cystic fibrosis (CF),emphysema radiation pneumonitis, and respiratory infection associatedwith inflammation caused by smoking, pulmonary edema, pneumoconiosis,sarcoidiosis, silicosis, asbestosis, berylliosis, coal worker'spneumonoconiosis (CWP), byssinosis, interstitial lung diseases (ILD)such as idiopathic pulmonary fibrosis, ILD associated with collagenvascular disorders, systemic lupus erythematosus, rheumatoid arthritis,ankylosing spondylitis, systemic sclerosis, and pulmonary inflammationthat is a result of or is secondary to another disorder such asinfluenza.

The present invention is not limited by the particular formulation of acomposition comprising a nanoemulsion of the present invention. Indeed,a composition comprising a nanoemulsion of the present invention maycomprise one or more different agents in addition to the nanoemulsion.These agents or cofactors include, but are not limited to, adjuvants,surfactants, additives, buffers, solubilizers, chelators, oils, salts,therapeutic agents, drugs, bioactive agents, antibacterials, andantimicrobial agents (e.g., antibiotics, antivirals, etc.). In someembodiments, a composition comprising a nanoemulsion of the presentinvention comprises an agent and/or co-factor that enhance the abilityof the nanoemulsion to kill a microbe (e.g., located in the respiratorytract). In some preferred embodiments, the presence of one or moreco-factors or agents reduces the amount of nanoemulsion required forkilling and/or attenuation of growth of a microbe. The present inventionis not limited by the type of co-factor or agent used in a therapeuticagent of the present invention.

In some embodiments, a co-factor or agent used in a nanoemulsioncomposition is a bioactive agent. For example, in some embodiments, thebioactive agent may be a bioactive agent useful in a cell (e.g., a cellexpressing a CFTR). Bioactive agents, as used herein, include diagnosticagents such as radioactive labels and fluorescent labels. Bioactiveagents also include molecules affecting the metabolism of a cell (e.g.,a cell expressing a CFTR), including peptides, nucleic acids, and othernatural and synthetic drug molecules. Bioactive agents include, but arenot limited to, adrenergic agent; adrenocortical steroid; adrenocorticalsuppressant; alcohol deterrent; aldosterone antagonist; amino acid;ammonia detoxicant; anabolic; analeptic; analgesic; androgen;anesthesia, adjunct to; anesthetic; anorectic; antagonist; anteriorpituitary suppressant; anthelmintic; anti-acne agent; anti-adrenergic;anti-allergic; anti-amebic; anti-androgen; anti-anemic; anti-anginal;anti-anxiety; anti-arthritic; anti-asthmatic; anti-atherosclerotic;antibacterial; anticholelithic; anticholelithogenic; anticholinergic;anticoagulant; anticoccidal; anticonvulsant; antidepressant;antidiabetic; antidiarrheal; antidiuretic; antidote; anti-emetic;anti-epileptic; anti-estrogen; antifibrinolytic; antifungal;antiglaucoma agent; antihemophilic; antihemorrhagic; antihistamine;antihyperlipidemia; antihyperlipoproteinemic; antihypertensive;antihypotensive; anti-infective; anti-infective, topical;anti-inflammatory; antikeratinizing agent; antimalarial; antimicrobial;antimigraine; antimitotic; antimycotic, antinauseant, antineoplastic,antineutropenic, antiobessional agent; antiparasitic; antiparkinsonian;antiperistaltic, antipneumocystic; antiproliferative; antiprostatichypertrophy; antiprotozoal; antipruritic; antipsychotic; antirheumatic;antischistosomal; antiseborrheic; antisecretory; antispasmodic;antithrombotic; antitussive; anti-ulcerative; anti-urolithic; antiviral;appetite suppressant; benign prostatic hyperplasia therapy agent; bloodglucose regulator; bone resorption inhibitor; bronchodilator; carbonicanhydrase inhibitor; cardiac depressant; cardioprotectant; cardiotonic;cardiovascular agent; choleretic; cholinergic; cholinergic agonist;cholinesterase deactivator; coccidiostat; cognition adjuvant; cognitionenhancer; depressant; diagnostic aid; diuretic; dopaminergic agent;ectoparasiticide; emetic; enzyme inhibitor; estrogen; fibrinolytic;fluorescent agent; free oxygen radical scavenger; gastrointestinalmotility effector; glucocorticoid; gonad-stimulating principle; hairgrowth stimulant; hemostatic; histamine H2 receptor antagonists;hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive;imaging agent; immunizing agent; immunomodulator; immunoregulator;immunostimulant; immunosuppressant; impotence therapy adjunct;inhibitor; keratolytic; LHRH agonist; liver disorder treatment;luteolysin; memory adjuvant; mental performance enhancer; moodregulator; mucolytic; mucosal protective agent; mydriatic; nasaldecongestant; neuromuscular blocking agent; neuroprotective; NMDAantagonist; non-hormonal sterol derivative; oxytocic; plasminogenactivator; platelet activating factor antagonist; platelet aggregationinhibitor; post-stroke and post-head trauma treatment; potentiator;progestin; prostaglandin; prostate growth inhibitor; prothyrotropin;psychotropic; pulmonary surface; radioactive agent; regulator; relaxant;repartitioning agent; scabicide; sclerosing agent; sedative;sedative-hypnotic; selective adenosine Al antagonist; serotoninantagonist; serotonin inhibitor; serotonin receptor antagonist; steroid;stimulant; suppressant; symptomatic multiple sclerosis; synergist;thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer;amyotrophic lateral sclerosis agent; cerebral ischemia agent; Paget'sdisease agent; unstable angina agent; uricosuric; vasoconstrictor;vasodilator; vulnerary; wound healing agent; xanthine oxidase inhibitor.

Molecules useful as antimicrobials can be delivered by the methods andcompositions of the invention, such that the respiratory infection isreduced or eliminated. Antibiotics that may find use inco-administration with a composition comprising a nanoemulsion of thepresent invention include, but are not limited to, agents or drugs thatare bactericidal and/or bacteriostatic (e.g., inhibiting replication ofbacteria or inhibiting synthesis of bacterial components required forsurvival of the infecting organism), including, but not limited to,almecillin, amdinocillin, amikacin, amoxicillin, amphomycin,amphotericin B, ampicillin, azacitidine, azaserine, azithromycin,azlocillin, aztreonam, bacampicillin, bacitracin, benzylpenicilloyl-polylysine, bleomycin, candicidin, capreomycin,carbenicillin, cefaclor, cefadroxil, cefamandole, cefazoline, cefdinir,cefepime, cefixime, cefinenoxime, cefinetazole, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin,cefpiramide, cefpodoxime, cefprozil, cefsulodin, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cephacetrile,cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin,cephradine, chloramphenicol, chlortetracycline, cilastatin, cinnamycin,ciprofloxacin, clarithromycin, clavulanic acid, clindamycin, clioquinol,cloxacillin, colistimethate, colistin, cyclacillin, cycloserine,cyclosporine, cyclo-(Leu-Pro), dactinomycin, dalbavancin, dalfopristin,daptomycin, daunorubicin, demeclocycline, detorubicin, dicloxacillin,dihydrostreptomycin, dirithromycin, doxorubicin, doxycycline,epirubicin, erythromycin, eveminomycin, floxacillin, fosfomycin, fusidicacid, gemifloxacin, gentamycin, gramicidin, griseofulvin, hetacillin,idarubicin, imipenem, iseganan, ivermectin, kanamycin, laspartomycin,linezolid, linocomycin, loracarbef, magainin, meclocycline, meropenem,methacycline, methicillin, mezlocillin, minocycline, mitomycin,moenomycin, moxalactam, moxifloxacin, mycophenolic acid, nafcillin,natamycin, neomycin, netilmicin, niphimycin, nitrofurantoin, novobiocin,oleandomycin, oritavancin, oxacillin, oxytetracycline, paromomycin,penicillamine, penicillin G, penicillin V, phenethicillin, piperacillin,plicamycin, polymyxin B, pristinamycin, quinupristin, rifabutin,rifampin, rifamycin, rolitetracycline, sisomicin, spectrinomycin,streptomycin, streptozocin, sulbactam, sultamicillin, tacrolimus,tazobactam, teicoplanin, telithromycin, tetracycline, ticarcillin,tigecycline, tobramycin, troleandomycin, tunicamycin, tyrthricin,vancomycin, vidarabine, viomycin, virginiamycin, BMS-284,756, L-749,345,ER-35,786, S-4661, L-786,392, MC-02479, PepS, RP 59500, and TD-6424.

In some embodiments, a composition comprising a nanoemulsion of thepresent invention comprises one or more mucoadhesives (See, e.g., U.S.Pat. App. No. 20050281843, hereby incorporated by reference in itsentirety). The present invention is not limited by the type ofmucoadhesive utilized. Indeed, a variety of mucoadhesives arecontemplated to be useful in the present invention including, but notlimited to, cross-linked derivatives of poly(acrylic acid) (e.g.,carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides (e.g., alginate and chitosan), hydroxypropylmethylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose.Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, use of amucoadhesive (e.g., in a composition comprising a nanoemulsion) enhanceskilling and/or attenuation of growth of a microbe (e.g., exposed to acomposition of the present invention) due to an increase in durationand/or amount of exposure to the nanoemulsion that a subject and/ormicrobe experiences when a mucoadhesive is used compared to the durationand/or amount of exposure to the nanoemulsion in the absence of usingthe mucoadhesive.

In some embodiments, a composition of the present invention may comprisesterile aqueous preparations. Acceptable vehicles and solvents include,but are not limited to, water, Ringer's solution, phosphate bufferedsaline and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed mineral or non-mineral oil maybe employed including synthetic mono-ordi-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.Carrier formulations suitable for mucosal, pulmonary, subcutaneous,intramuscular, intraperitoneal, intravenous, or administration via otherroutes may be found in Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa.

A composition comprising a nanoemulsion of the present invention can beused therapeutically (e.g., to kill and/or attenuate growth of anexisting infection) or as a prophylactic (e.g., to prevent microbialgrowth and/or colonization (e.g., to prevent signs or symptoms ofdisease)). A composition comprising a nanoemulsion of the presentinvention can be administered to a subject via a number of differentdelivery routes and methods.

For example, the compositions of the present invention can beadministered to a subject (e.g., mucosally or by pulmonary route) bymultiple methods, including, but not limited to: being suspended in asolution and applied to a surface; being suspended in a solution andsprayed onto a surface using a spray applicator; being mixed with amucoadhesive and applied (e.g., sprayed or wiped) onto a surface (e.g.,mucosal or pulmonary surface); being placed on or impregnated onto anasal and/or pulmonary applicator and applied; being applied by acontrolled-release mechanism; applied using a nebulizer, aerosolized,being applied as a liposome; or being applied on a polymer.

In some embodiments, compositions of the present invention areadministered mucosally (e.g., using standard techniques; See, e.g.,Remington: The Science and Practice of Pharmacy, Mack PublishingCompany, Easton, Pa., 19th edition, 1995 (e.g., for mucosal deliverytechniques, including intranasal and pulmonary techniques), as well asEuropean Publication No. 517,565 and Illum et al., J. Controlled Rel.,1994, 29:133-141 (e.g., for techniques of intranasal administration),each of which is hereby incorporated by reference in its entirety). Thepresent invention is not limited by the route of administration.

Methods of intranasal and pulmonary administration are well known in theart, including the administration of a droplet or spray form of thenanoemulsion into the nasopharynx of a subject to be treated. In someembodiments, a nebulized or aerosolized composition comprising ananoemulsion is provided. Enteric formulations such as gastro resistantcapsules for oral administration, suppositories for rectal or vaginaladministration may also form part of this invention. Compositions of thepresent invention may also be administered via the oral route. Underthese circumstances, a composition comprising a nanoemulsion maycomprise a pharmaceutically acceptable excipient and/or include alkalinebuffers, or enteric capsules. Formulations for nasal delivery mayinclude those with dextran or cyclodextran and saponin as an adjuvant.

In preferred embodiments, a nanoemulsion of the present invention isadministered via a pulmonary delivery route and/or means. In someembodiments, an aqueous solution containing the nanoemulsion is gentlyand thoroughly mixed to form a solution. The solution is sterilefiltered (e.g., through a 0.2 micron filter) into a sterile, enclosedvessel. Under sterile conditions, the solution is passed through anappropriately small orifice to make droplets (e.g., between 0.1 and 10microns).

The particles may be administered using any of a number of differentapplicators. Suitable methods for manufacture and administration aredescribed in the following U.S. Pat. Nos. 6,592,904; 6,518,239;6,423,344; 6,294,204; 6,051,256 and 5,997,848 to INHALE (now NEKTAR);and U.S. Pat. No. 5,985,309; RE37,053; U.S. Pat. Nos. 6,436,443;6,447,753; 6,503,480; and 6,635,283, to Edwards, et al. (MIT, AIR), eachof which is hereby incorporated

Thus, in some embodiments, compositions of the present invention areadministered by pulmonary delivery. For example, a composition of thepresent invention can be delivered to the lungs of a subject (e.g., ahuman) via inhalation (See, e.g., Adjei, et al. Pharmaceutical Research1990; 7:565-569; Adjei, et al. Int. J. Pharmaceutics 1990; 63:135-144;Braquet, et al. J. Cardiovascular Pharmacology 1989 143-146; Hubbard, etal. (1989) Annals of Internal Medicine, Vol. III, pp. 206-212; Smith, etal. J. Clin. Invest. 1989; 84:1145-1146; Oswein, et al. “Aerosolizationof Proteins”, 1990; Proceedings of Symposium on Respiratory DrugDelivery II Keystone, Colorado; Debs, et al. J. Immunol. 1988;140:3482-3488; and U.S. Pat. No. 5,284,656 to Platz, et al, each ofwhich are hereby incorporated by reference in its entirety). A methodand composition for pulmonary delivery of drugs for systemic effect isdescribed in U.S. Pat. No. 5,451,569 to Wong, et al., herebyincorporated by reference; See also U.S. Pat. No. 6,651,655 to Licalsiet al., hereby incorporated by reference in its entirety)). In someembodiments, a composition comprising a nanoemulsion is administered toa subject by more than one route or means (e.g., administered viapulmonary route as well as a mucosal route).

Further contemplated for use in the practice of this invention are awide range of mechanical devices designed for pulmonary and/or nasalmucosal delivery of pharmaceutical agents including, but not limited to,nebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices suitable for the practice of thisinvention are the ULTRAVENT nebulizer (Mallinckrodt Inc., St. Louis,Mo.); the ACORN II nebulizer (Marquest Medical Products, Englewood,Colo.); the VENTOLIN metered dose inhaler (Glaxo Inc., Research TrianglePark, N.C.); and the SPINHALER powder inhaler (Fisons Corp., Bedford,Mass.). All such devices require the use of formulations suitable fordispensing of the therapeutic agent. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants, surfactants, carriers and/or other agents useful in therapy.Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Thus, in some embodiments, a composition comprising a nanoemulsion ofthe present invention may be used to protect and/or treat a subjectsusceptible to, or suffering from, a disease by means of administeringcompositions comprising a nanoemulsion by mucosal, intramuscular,intraperitoneal, intradermal, transdermal, pulmonary, intravenous,subcutaneous or other route of administration described herein. Methodsof systemic administration of the nanoemulsion and/or agentco-administered with the nanoemulsion may include conventional syringesand needles, or devices designed for ballistic delivery (See, e.g., WO99/27961, hereby incorporated by reference), or needleless pressureliquid jet device (See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No.5,993,412, each of which are hereby incorporated by reference), ortransdermal patches (See, e.g., WO 97/48440; WO 98/28037, each of whichare hereby incorporated by reference). In some embodiments, the presentinvention provides a delivery device for systemic administration,pre-filled with the nanoemulsion composition of the present invention.

As described above, the present invention is not limited by the type ofsubject administered a composition of the present invention. Indeed, awide variety of subjects are contemplated to be benefited fromadministration of a composition of the present invention. In preferredembodiments, the subject is a human. In some embodiments, human subjectsare of any age (e.g., adults, children, infants, etc.) that have been orare likely to become exposed to a microorganism. In some embodiments,the human subjects are subjects that are more likely to receive a directexposure to pathogenic microorganisms or that are more likely to displaysigns and symptoms of disease after exposure to a pathogen (e.g.,subjects with CF or asthma, subjects in the armed forces, governmentemployees, frequent travelers, persons attending or working in a schoolor daycare, health care workers, an elderly person, an immunocompromisedperson, and emergency service employees (e.g., police, fire, EMTemployees)). In some embodiments, any one or all members of the generalpublic can be administered a composition of the present invention (e.g.,to prevent the occurrence or spread of disease). For example, in someembodiments, compositions and methods of the present invention areutilized to treat a group of people (e.g., a population of a region,city, state and/or country) for their own health (e.g., to prevent ortreat disease) and/or to prevent or reduce the risk of disease spreadfrom animals (e.g., birds, cattle, sheep, pigs, etc.) to humans. In someembodiments, the subjects are non-human mammals (e.g., pigs, cattle,goats, horses, sheep, or other livestock; or mice, rats, rabbits orother animal). In some embodiments, compositions and methods of thepresent invention are utilized in research settings (e.g., with researchanimals).

A composition comprising a nanoemulsion of the present invention can beadministered (e.g., to a subject (e.g., via pulmonary and/or mucosalroute) or to microbes (e.g., bacteria (e.g., opportunistic and/orpathogenic bacteria (e.g., residing on or within the respiratory systemof a subject and/or on or within a burn wound)))) as a therapeutic or asa prophylactic to prevent microbial infection. Thus, in someembodiments, the present invention provides a method of alteringmicrobial (e.g., bacterial (e.g., opportunistic and/or pathogenicbacterial) growth comprising administering a composition comprising ananoemulsion to the microbes (e.g., bacteria (e.g., opportunistic and/orpathogenic bacteria). In some embodiments, administration of acomposition comprising a nanoemulsion to the microbes (e.g., bacteria(e.g., opportunistic and/or pathogenic bacteria) kills the microbes. Insome embodiments, administration of a composition comprisingnanoemulsion to the microbes (e.g., bacteria (e.g., opportunistic and/orpathogenic bacteria) inhibits growth of the microbes. It is contemplatedthat a composition comprising a nanoemulsion can be administered tomicrobes (e.g., bacteria (e.g., opportunistic and/or pathogenic bacteria(e.g., residing within the respiratory tract))) via a number of deliveryroutes and/or mechanisms.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, preferablydo not unduly interfere with the biological activities of the componentsof the compositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like) that do not deleteriouslyinteract with the nanoemulsion. In some embodiments, nanoemulsioncompositions of the present invention are administered in the form of apharmaceutically acceptable salt. When used the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof. Such salts include, but are not limited to, those prepared fromthe following acids: hydrochloric, hydrobromic, sulphuric, nitric,phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric,citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include, but are not limited to, acetic acidand a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid anda salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).Suitable preservatives may include benzalkonium chloride (0.003-0.03%w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) andthimerosal (0.004-0.02% w/v).

In some embodiments, a composition comprising a nanoemulsion isco-administered with one or more antibiotics. For example, one or moreantibiotics may be administered with, before and/or after administrationof a composition comprising a nanoemulsion. The present invention is notlimited by the type of antibiotic co-administered. Indeed, a variety ofantibiotics may be co-administered including, but not limited to,ρ-lactam antibiotics, penicillins (such as natural penicillins,aminopenicillins, penicillinase-resistant penicillins, carboxypenicillins, ureido penicillins), cephalosporins (first generation,second generation, and third generation cephalosporins), and otherβ-lactams (such as imipenem, monobactams,), β-lactamase inhibitors,vancomycin, aminoglycosides and spectinomycin, tetracyclines,chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin,metronidazole, polymyxins, doxycycline, quinolones (e.g.,ciprofloxacin), sulfonamides, trimethoprim, and quinolines.

A wide variety of antimicrobial agents are currently available for usein treating bacterial, fungal and viral infections. For a comprehensivetreatise on the general classes of such drugs and their mechanisms ofaction, the skilled artisan is referred to Goodman & Gilman's “ThePharmacological Basis of Therapeutics” Eds. Hardman et al., 9th Edition,Pub. McGraw Hill, chapters 43 through 50, 1996, (herein incorporated byreference in its entirety). Generally, these agents include agents thatinhibit cell wall synthesis (e.g., penicillins, cephalosporins,cycloserine, vancomycin, bacitracin); and the imidazole antifungalagents (e.g., miconazole, ketoconazole and clotrimazole); agents thatact directly to disrupt the cell membrane of the microorganism (e.g.,detergents such as polmyxin and colistimethate and the antifungalsnystatin and amphotericin B); agents that affect the ribosomal subunitsto inhibit protein synthesis (e.g., chloramphenicol, the tetracyclines,erthromycin and clindamycin); agents that alter protein synthesis andlead to cell death (e.g., aminoglycosides); agents that affect nucleicacid metabolism (e.g., the rifamycins and the quinolones); theantimetabolites (e.g., trimethoprim and sulfonamides); and the nucleicacid analogues such as zidovudine, gangcyclovir, vidarabine, andacyclovir which act to inhibit viral enzymes essential for DNAsynthesis. Various combinations of antimicrobials may be employed.

The present invention also includes methods involving co-administrationof a composition comprising a nanoemulsion with one or more additionalactive and/or anti-infective agents. In co-administration procedures,the agents may be administered concurrently or sequentially. In oneembodiment, the compositions described herein are administered prior tothe other active agent(s). The pharmaceutical formulations and modes ofadministration may be any of those described herein. In addition, thetwo or more co-administered agents may each be administered usingdifferent modes (e.g., routes) or different formulations. The additionalagents to be co-administered (e.g., antibiotics, a second type ofnanoemulsion, etc.) can be any of the well-known agents in the art,including, but not limited to, those that are currently in clinical use.

In some embodiments, a composition comprising a nanoemulsion isadministered to a subject via more than one route. For example, asubject may benefit from receiving mucosal administration (e.g., nasaladministration or other mucosal routes described herein) and,additionally, receiving one or more other routes of administration(e.g., pulmonary administration (e.g., via a nebulizer, inhaler, orother methods described herein.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions, increasing convenience to thesubject and a physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer based systems such as poly (lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109,hereby incorporated by reference. Delivery systems also includenon-polymer systems that are: lipids including sterols such ascholesterol, cholesterol esters and fatty acids or neutral fats such asmono-di- and tri-glycerides; hydrogel release systems; sylastic systems;peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5,736,152, each of which is hereby incorporated by reference and (b)diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,854,480, 5,133,974 and 5,407,686, each of which is hereby incorporatedby reference. In addition, pump-based hardware delivery systems can beused, some of which are adapted for implantation.

In preferred embodiments, a composition comprising a nanoemulsion of thepresent invention comprises a suitable amount of the nanoemulsion tokill and/or attenuate growth of microbes (e.g., pathogenic microbes(e.g., pathogenic bacteria, viruses, etc.)) in a subject whenadministered to the subject. The present invention is not limited by theamount of nanoemulsion used. In some preferred embodiments, the amountof nanoemulsion in a composition comprising a nanoemulsion is selectedas that amount which kills and/or attenuates microbial growth withoutsignificant, adverse side effects. The amount will vary depending uponwhich specific nanoemulsion(s) is/are employed, and can vary fromsubject to subject, depending on a number of factors including, but notlimited to, the species, age and general condition (e.g., health) of thesubject, and the mode of administration. Procedures for determining theappropriate amount of nanoemulsion administered to a subject to killand/or attenuate growth of a microbe (e.g., pathogenic microbe (e.g.,pathogenic bacteria, viruses, etc.)) in a subject can be readilydetermined using known means by one of ordinary skill in the art.

In some embodiments, it is expected that each dose (e.g., of acomposition comprising a nanoemulsion (e.g., administered to a subjectto kill and/or attenuate microbial growth)) comprises 10-40%nanoemulsion, in some embodiments, 20% nanoemulsion, in some embodimentsless than 20% (e.g., 15%, 10%, 8%, 5% or less nanoemulsion), and in someembodiments greater than 20% nanoemulsion (e.g., 25%, 30%, 35%, 40% ormore nanoemulsion). An optimal amount for a particular administration(e.g., to kill and/or attenuate microbial growth) can be ascertained byone of skill in the art using standard studies involving observation ofmicrobial growth and/or death and other responses in subjects.

In some embodiments, it is expected that each dose (e.g., of acomposition comprising a nanoemulsion (e.g., administered to a subjectto kill and/or attenuate microbial growth)) is from 0.001 to 40% or more(e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15%, 20%, 30%, 40% or more)by weight nanoemulsion.

Similarly, the present invention is not limited by the duration of timea nanoemulsion is administered to a subject (e.g., to kill and/orattenuate microbial growth). In some embodiments, a nanoemulsion isadministered one or more times (e.g. twice, three times, four times ormore) daily. In some embodiments, a composition comprising ananoemulsion is administered one or more times a day until an infectionis eradicated or microbial growth and/or presence has been reduced to adesired level. In some embodiments, a composition comprising ananoemulsion of the present invention is formulated in a concentrateddose that can be diluted prior to administration to a subject. Forexample, dilutions of a concentrated composition may be administered toa subject such that the subject receives any one or more of the specificdosages provided herein. In some embodiments, dilution of a concentratedcomposition may be made such that a subject is administered (e.g., in asingle dose) a composition comprising 0.5-50% of the nanoemulsionpresent in the concentrated composition. Concentrated compositions arecontemplated to be useful in a setting in which large numbers ofsubjects may be administered a composition of the present invention(e.g., a hospital). In some embodiments, a composition comprising ananoemulsion of the present invention (e.g., a concentrated composition)is stable at room temperature for more than 1 week, in some embodimentsfor more than 2 weeks, in some embodiments for more than 3 weeks, insome embodiments for more than 4 weeks, in some embodiments for morethan 5 weeks, and in some embodiments for more than 6 weeks.

Dosage units may be proportionately increased or decreased based onseveral factors including, but not limited to, the weight, age, andhealth status of the subject. In addition, dosage units may be increasedor decreased for subsequent administrations.

In some embodiments, a composition comprising a nanoemulsion isadministered to a subject under conditions such that microbes (e.g.,bacteria (e.g., opportunistic and/or pathogenic bacteria)) are killed.In some embodiments, a composition comprising a nanoemulsion isadministered to a subject under conditions such that microbial (e.g.,bacterial (e.g., opportunistic and/or pathogenic bacterial) growth isprohibited and/or attenuated. In some embodiments, greater than 90%(e.g., greater than 95%, 98%, 99%, all detectable) of microbes (e.g.,bacteria (e.g., opportunistic and/or pathogenic bacteria) are killed. Insome embodiments, there is greater than 2 log (e.g., greater than 3 log,4 log, 5 log, or more) reduction in microbe (e.g., bacteria (e.g.,opportunistic and/or pathogenic bacteria) presence. In some embodiments,reduction and/or killing is observed in one hour or less (e.g., 45minutes, 30 minutes, 15 minutes, or less). In some embodiments,reduction and/or killing is observed in 6 hours or less (e.g., 5 hours,4, hours, 3 hours, two hours or less than one hour). In someembodiments, reduction and/or killing is observed in two days or lessfollowing initial treatment (e.g., less than 24 hours, less than 20hours, 18 hours or less). In some embodiments, the reduction and/orkilling is observed in three days or less, four days or less, or fivedays or less.

A composition comprising a nanoemulsion of the present invention findsuse where the nature of the infectious and/or disease causing agent(e.g., causing signs, symptoms or indications of respiratory infection)is known, as well as where the nature of the infectious and/or diseasecausing agent is unknown (e.g., in emerging disease (e.g., of pandemicproportion (e.g., influenza or other outbreaks of disease))). Forexample, the present invention contemplates use of the compositions ofthe present invention in treatment of or prevention of infectionsassociated with an emergent infectious and/or disease causing agent yetto be identified (e.g., isolated and/or cultured from a diseased personbut without genetic, biochemical or other characterization of theinfectious and/or disease causing agent).

It is contemplated that the compositions and methods of the presentinvention will find use in various settings, including researchsettings. For example, compositions and methods of the present inventionalso find use in studies of the immune system (e.g., characterization ofadaptive immune responses (e.g., protective immune responses (e.g.,mucosal or systemic immunity))). Uses of the compositions and methodsprovided by the present invention encompass human and non-human subjectsand samples from those subjects, and also encompass researchapplications using these subjects. Compositions and methods of thepresent invention are also useful in studying and optimizingnanoemulsions, immunogens, and other components and for screening fornew components. Thus, it is not intended that the present invention belimited to any particular subject and/or application setting.

The formulations can be tested in vivo in a number of animal modelsdeveloped for the study of pulmonary, mucosal and other routes ofdelivery. As is readily apparent, the compositions of the presentinvention are useful for preventing and/or treating a wide variety ofdiseases and infections caused by viruses, bacteria, parasites, andfungi. Not only can the compositions be used prophylactically ortherapeutically, as described above, the compositions can also be usedin order to prepare antibodies, both polyclonal and monoclonal (e.g.,for diagnostic purposes), as well as for immunopurification of anantigen of interest.

In some embodiments, the present invention provides a kit comprising acomposition comprising a nanoemulsion. In some embodiments, the kitfurther provides a device for administering the composition. The presentinvention is not limited by the type of device included in the kit. Insome embodiments, the device is configured for pulmonary application ofthe composition of the present invention (e.g., a nasal inhaler or nasalmister). In some embodiments, a kit comprises a composition comprising ananoemulsion in a concentrated form (e.g., that can be diluted prior toadministration to a subject).

In some embodiments, all kit components are present within a singlecontainer (e.g., vial or tube). In some embodiments, each kit componentis located in a single container (e.g., vial or tube). In someembodiments, one or more kit components are located in a singlecontainer (e.g., vial or tube) with other components of the same kitbeing located in a separate container (e.g., vial or tube). In someembodiments, a kit comprises a buffer. In some embodiments, the kitfurther comprises instructions for use.

Therapeutic and Prophylactic Use of Immunogenic Compositions

The present invention provides immunogenic nanoemulsion compositions andmethods of using the same for the induction of immune responses (e.g.,innate and/or adaptive immune responses (e.g., for generation of hostimmunity against a bacterial species of the genus Burkholderia (e.g., B.cenocepacia, B. multivorans, etc.))). Compositions and methods of thepresent invention find use in, among other things, clinical (e.g.therapeutic and preventative medicine (e.g., vaccination)) and researchapplications.

As shown in Example 10, an immunogenic composition (e.g., comprising ananoemulsion and Bukholderia antigen) of the present invention induces(e.g., when administered to a subject) both systemic and mucosalimmunity. Thus, in some preferred embodiments, administration of acomposition comprising a nanoemulsion and Burkholderia antigen to asubject results in protection against an exposure (e.g., a mucosaland/or respiratory exposure) to Burkholderia. Although an understandingof the mechanism is not necessary to practice the present invention andthe present invention is not limited to any particular mechanism ofaction, mucosal administration (e.g., vaccination) provides protectionagainst Burkholderia (e.g., Burkholderia cepacia) infection (e.g., thatinitiates at a mucosal surface). Although it has heretofore provendifficult to stimulate secretory IgA responses and protection againstpathogens that invade at mucosal surfaces (See, e.g., Mestecky et al,Mucosal Immunology. 3ed edn. (Academic Press, San Diego, 2005)), in someembodiments, the present invention provides compositions and methods forstimulating mucosal immunity (e.g., a protective IgA response) towards apathogen (e.g., pathogenic species of Burkholderia, Haemophilus,Staphylococcus or other bacterial genus) in a subject.

In some embodiments, the present invention provides a composition (e.g.,a composition comprising a nanoemulsion and Burkholderia (e.g.,Burkholderia cenocepacia) antigen (e.g., an immunogenic polypeptidecomprising an amino acid sequence of SEQ ID NO. 1 or SEQ ID NOS. 2-16))to serve as a mucosal vaccine. In some embodiments, an immunogeniccomposition comprising nanoemulsion (NE) and Burkholderia antigen isproduced with NE and killed whole cell bacteria of the genusBurkholderia (e.g., Burkholderia cepacia (e.g., killed usingnanoemulsion, alcohol (e.g., ethanol), or other methods), isolated,purified and/or recombinant protein and/or saccharide component ofBurkholderia (e.g., protein/peptide (e.g., Burkholderia-derived protein,live-virus-vector-derived protein, recombinant protein, recombinantdenatured protein/antigens, small peptide segments protein/antigen).

In some preferred embodiments, the present invention provides acomposition for generating an immune response comprising a NE and animmunogen (e.g., a purified, isolated or synthetic Burkholderia proteinor derivative, variant, or analogue thereof; or, one or more species ofBurkholderia (e.g., Burkholderia multivorans (e.g., killed and orinactivated whole cell bacteria). When administered to a subject, acomposition of the present invention stimulates an immune responseagainst the immunogen within the subject. Although an understanding ofthe mechanism is not necessary to practice the present invention and thepresent invention is not limited to any particular mechanism of action,in some embodiments, generation of an immune response (e.g., resultingfrom administration of a composition comprising a nanoemulsion and animmunogen) provides total or partial immunity to the subject (e.g., fromsigns, symptoms or conditions of a disease associated with Burkholderiainfection (e.g., strep throat, meningitis, bacterial pneumoniae,endocarditis, erysipelas and/or necrotizing fasciitis)). Without beingbound to any specific theory, protection and/or immunity from disease(e.g., the ability of a subject's immune system to prevent or attenuate(e.g., suppress) a sign, symptom or condition of disease) after exposureto an immunogenic composition of the present invention is due toadaptive (e.g., acquired) immune responses (e.g., immune responsesmediated by B and T cells following exposure to a NE comprising animmunogen of the present invention (e.g., immune responses that exhibitincreased specificity and reactivity towards Burkholderia (e.g.,Burkholderia cepacia)). Thus, in some embodiments, the compositions andmethods of the present invention are used prophylactically ortherapeutically to prevent or attenuate a sign, symptom or conditionassociated with Burkholderia (e.g., Burkholderia cepacia)).

In some embodiments, a NE comprising an immunogen (e.g., a Burkholderia(e.g., Burkholderia cepacia) antigen) is administered alone. In someembodiments, a composition comprising a NE and an immunogen (e.g., aBurkholderia (e.g., Burkholderia cepacia) antigen) comprises one or moreother agents (e.g., a pharmaceutically acceptable carrier, adjuvant,excipient, and the like). In some embodiments, a composition forstimulating an immune response of the present invention is administeredin a manner to induce a humoral immune response. In some embodiments, acomposition for stimulating an immune response of the present inventionis administered in a manner to induce a cellular (e.g., cytotoxic Tlymphocyte) immune response, rather than a humoral response. In someembodiments, a composition comprising a NE and an immunogen of thepresent invention induces both a cellular and humoral immune response.

The present invention is not limited by the isotype, strain or speciesof Burkholderia (e.g., Burkholderia cepacia) used in a compositioncomprising a NE and immunogen. Indeed, each Burkholderia (e.g.,Burkholderia cepacia) family member alone, or in combination withanother family member, may be used to generate a composition comprisinga NE and an immunogen (e.g., used to generate an immune response) of thepresent invention. Exemplary species of Burkholderia are describedherein.

In some embodiments, the Burkholderia (e.g., Burkholderia cepacia)species utilized is a modified (e.g., genetically modified (e.g.,naturally modified via natural selection or modified using recombinantgenetic techniques)) species that displays greater pathogenic capacity(e.g., causes more sever Burkholderia—(e.g., Burkholderiacepacia)-induced disease (e.g., comprising enhanced and/or more severerespiratory infection, etc.)). In some embodiments, any one or moremembers of the genus Burkholderia is utilized in an immunoreactivecomposition of the invention including but not limited to B.cenocepacia, B. dolosa, B. multivorans, B. ambifaria, B. vietnamiensis,B. ubonensis, B. thailandensis, B. graminis, B. oklahomensis, B.pseudomallei, B. xenovorans, B. phytofirmans, B. phymatum, R.metallidurans, R. eutropha, R. solanacearum.

The present invention is not limited by the Burkholderia strain used.Indeed, a variety of Burkholderia strains are contemplated to be usefulin the present invention including, but not limited to, classicalstrains, attenuated strains, non-replicating strains, modified strains(e.g., genetically or mechanically modified strains (e.g., to becomemore or less virulent)), or other serially diluted strains ofBurkholderia. A composition comprising a NE and immunogen may compriseone or more strains of Burkholderia cenocepacia and/or other type ofBurkholderia (e.g., Burkholderia multivorans). Additionally, acomposition comprising a NE and immunogen may comprise one or morestrains of Burkholderia and, in addition, one or more strains of anon-Burkholderia immunogen.

In some embodiments, the immunogen may comprise one or more antigensderived from a pathogen (e.g., Burkholderia). For example, in someembodiments, the immunogen is a purified, recombinant, synthetic, orotherwise isolated protein (e.g., added to the NE to generate animmunogenic composition). Similarly, the immunogenic protein may be aderivative, analogue or otherwise modified (e.g., conjugated) form of aprotein from a pathogen.

The present invention is not limited by the particular formulation of acomposition comprising a NE and immunogen of the present invention.Indeed, a composition comprising a NE and immunogen of the presentinvention may comprise one or more different agents in addition to theNE and immunogen. These agents or cofactors include, but are not limitedto, adjuvants, surfactants, additives, buffers, solubilizers, chelators,oils, salts, therapeutic agents, drugs, bioactive agents,antibacterials, and antimicrobial agents (e.g., antibiotics, antivirals,etc.). In some embodiments, a composition comprising a NE and immunogenof the present invention comprises an agent and/or co-factor thatenhance the ability of the immunogen to induce an immune response (e.g.,an adjuvant). In some preferred embodiments, the presence of one or moreco-factors or agents reduces the amount of immunogen required forinduction of an immune response (e.g., a protective immune response(e.g., protective immunization)). In some embodiments, the presence ofone or more co-factors or agents can be used to skew the immune responsetowards a cellular (e.g., T cell mediated) or humoral (e.g., antibodymediated) immune response. The present invention is not limited by thetype of co-factor or agent used in a therapeutic agent of the presentinvention.

Adjuvants are described in general in Vaccine Design—the Subunit andAdjuvant Approach, edited by Powell and Newman, Plenum Press, New York,1995. The present invention is not limited by the type of adjuvantutilized (e.g., for use in a composition (e.g., pharmaceuticalcomposition) comprising a NE and immunogen). For example, in someembodiments, suitable adjuvants include an aluminum salt such asaluminum hydroxide gel (alum) or aluminum phosphate. In someembodiments, an adjuvant may be a salt of calcium, iron or zinc, or maybe an insoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatised polysaccharides, orpolyphosphazenes.

In some embodiments, it is preferred that a composition comprising a NEand immunogen of the present invention comprises one or more adjuvantsthat induce a Th1-type response. However, in other embodiments, it willbe preferred that a composition comprising a NE and immunogen of thepresent invention comprises one or more adjuvants that induce a Th2-typeresponse.

In general, an immune response is generated to an antigen through theinteraction of the antigen with the cells of the immune system. Immuneresponses may be broadly categorized into two categories: humoral andcell mediated immune responses (e.g., traditionally characterized byantibody and cellular effector mechanisms of protection, respectively).These categories of response have been termed Th1-type responses(cell-mediated response), and Th2-type immune responses (humoralresponse).

Stimulation of an immune response can result from a direct or indirectresponse of a cell or component of the immune system to an intervention(e.g., exposure to an immunogen). Immune responses can be measured inmany ways including activation, proliferation or differentiation ofcells of the immune system (e.g., B cells, T cells, dendritic cells,APCs, macrophages, NK cells, NKT cells etc.); up-regulated ordown-regulated expression of markers and cytokines; stimulation of IgA,IgM, or IgG titer; splenomegaly (including increased spleencellularity); hyperplasia and mixed cellular infiltrates in variousorgans. Other responses, cells, and components of the immune system thatcan be assessed with respect to immune stimulation are known in the art.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, compositions andmethods of the present invention induce expression and secretion ofcytokines (e.g., by macrophages, dendritic cells and CD4+ T cells).Modulation of expression of a particular cytokine can occur locally orsystemically. It is known that cytokine profiles can determine T cellregulatory and effector functions in immune responses. In someembodiments, Th1-type cytokines can be induced, and thus, theimmunostimulatory compositions of the present invention can promote aTh1 type antigen-specific immune response including cytotoxic T-cells(e.g., thereby avoiding unwanted Th2 type immune responses (e.g.,generation of Th2 type cytokines (e.g., IL-13) involved in enhancing theseverity of disease (e.g., IL-13 induction of mucus formation))).

Cytokines play a role in directing the T cell response. Helper (CD4+) Tcells orchestrate the immune response of mammals through production ofsoluble factors that act on other immune system cells, including B andother T cells. Most mature CD4+ T helper cells express one of twocytokine profiles: Th1 or Th2. Th1-type CD4+ T cells secrete IL-2, IL-3,IFN-γ, GM-CSF and high levels of TNF-α. Th2 cells express IL-3, IL-4,IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. Th1 typecytokines promote both cell-mediated immunity, and humoral immunity thatis characterized by immunoglobulin class switching to IgG2a in mice andIgG1 in humans. Th1 responses may also be associated with delayed-typehypersensitivity and autoimmune disease. Th2 type cytokines induceprimarily humoral immunity and induce class switching to IgG1 and IgE.The antibody isotypes associated with Th1 responses generally haveneutralizing and opsonizing capabilities whereas those associated withTh2 responses are associated more with allergic responses.

Several factors have been shown to influence skewing of an immuneresponse towards either a Th1 or Th2 type response. The bestcharacterized regulators are cytokines IL-12 and IFN-γ are positive Th1and negative Th2 regulators. IL-12 promotes IFN-γ production, and IFN-γprovides positive feedback for IL-12. IL-4 and IL-10 appear importantfor the establishment of the Th2 cytokine profile and to down-regulateTh1 cytokine production.

Thus, in preferred embodiments, the present invention provides a methodof stimulating a Th1-type immune response in a subject comprisingadministering to a subject a composition comprising a NE and animmunogen. However, in other embodiments, the present invention providesa method of stimulating a Th2-type immune response in a subject (e.g.,if balancing of a T cell mediated response is desired) comprisingadministering to a subject a composition comprising a NE and animmunogen. In further preferred embodiments, adjuvants can be used(e.g., can be co-administered with a composition of the presentinvention) to skew an immune response toward either a Th1 or Th2 typeimmune response. For example, adjuvants that induce Th2 or weak Th1responses include, but are not limited to, alum, saponins, and SB-As4.Adjuvants that induce Th1 responses include but are not limited to MPL,MDP, ISCOMS, IL-12, IFN-γ, and SB-AS2.

Several other types of Th1-type immunogens can be used (e.g., as anadjuvant) in compositions and methods of the present invention. Theseinclude, but are not limited to, the following. In some embodiments,monophosphoryl lipid A (e.g., in particular 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL)), is used. 3D-MPL is a well knownadjuvant manufactured by Ribi Immunochem, Mont. Chemically it is oftensupplied as a mixture of 3-de-O-acylated monophosphoryl lipid A witheither 4, 5, or 6 acylated chains. In some embodiments, diphosphoryllipid A, and 3-O-deacylated variants thereof are used. Each of theseimmunogens can be purified and prepared by methods described in GB2122204B, hereby incorporated by reference in its entirety. Otherpurified and synthetic lipopolysaccharides have been described (See,e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al., 1986,Int. Arch. Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987,Immunology, 60(1):141-6; and EP 0 549 074, each of which is herebyincorporated by reference in its entirety). In some embodiments, 3D-MPLis used in the form of a particulate formulation (e.g., having a smallparticle size less than 0.2 μm in diameter, described in EP 0 689 454,hereby incorporated by reference in its entirety).

In some embodiments, saponins are used as an immunogen (e.g., Th1-typeadjuvant) in a composition of the present invention. Saponins are wellknown adjuvants (See, e.g., Lacaille-Dubois and Wagner (1996)Phytomedicine vol 2 pp 363-386). Examples of saponins include Quil A(derived from the bark of the South American tree Quillaja SaponariaMolina), and fractions thereof (See, e.g., U.S. Pat. No. 5,057,540;Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0362 279, each of which is hereby incorporated by reference in itsentirety). Also contemplated to be useful in the present invention arethe haemolytic saponins QS7, QS 17, and QS21 (HPLC purified fractions ofQuil A; See, e.g., Kensil et al. (1991). J. Immunology 146, 431-437,U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0 362 279, eachof which is hereby incorporated by reference in its entirety). Alsocontemplated to be useful are combinations of QS21 and polysorbate orcyclodextrin (See, e.g., WO 99/10008, hereby incorporated by referencein its entirety.

In some embodiments, an immunogenic oligonucleotide containingunmethylated CpG dinucleotides (“CpG”) is used as an adjuvant in thepresent invention. CpG is an abbreviation for cytosine-guanosinedinucleotide motifs present in DNA. CpG is known in the art as being anadjuvant when administered by both systemic and mucosal routes (See,e.g., WO 96/02555, EP 468520, Davis et al., J. Immunol, 1998,160(2):870-876; McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6;and U.S. Pat. App. No. 20050238660, each of which is hereby incorporatedby reference in its entirety). For example, in some embodiments, theimmunostimulatory sequence is Purine-Purine-C-G-pyrimidine-pyrimidine;wherein the CG motif is not methylated.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, the presence of oneor more CpG oligonucleotides activate various immune subsets includingnatural killer cells (which produce IFN-γ) and macrophages. In someembodiments, CpG oligonucleotides are formulated into a composition ofthe present invention for inducing an immune response. In someembodiments, a free solution of CpG is co-administered together with anantigen (e.g., present within a NE solution (See, e.g., WO 96/02555;hereby incorporated by reference). In some embodiments, a CpGoligonucleotide is covalently conjugated to an antigen (See, e.g., WO98/16247, hereby incorporated by reference), or formulated with acarrier such as aluminium hydroxide (See, e.g., Brazolot-Millan et al.,Proc. Natl. Acad Sci., USA, 1998, 95(26), 15553-8).

In some embodiments, adjuvants such as Complete Freunds Adjuvant andIncomplete Freunds Adjuvant, cytokines (e.g., interleukins (e.g., IL-2,IFN-γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosisfactor, etc.), detoxified mutants of a bacterial ADP-ribosylating toxinsuch as a cholera toxin (CT), a pertussis toxin (PT), or an E. Coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S 109(where serine is substituted for the wild-type amino acid at position109), and PT-K9/G129 (where lysine is substituted for the wild-typeamino acid at position 9 and glycine substituted at position 129) (See,e.g., WO93/13202 and WO92/19265, each of which is hereby incorporated byreference), and other immunogenic substances (e.g., that enhance theeffectiveness of a composition of the present invention) are used with acomposition comprising a NE and immunogen of the present invention.

Additional examples of adjuvants that find use in the present inventioninclude poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; VirusResearch Institute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); and Leishmania elongation factor (apurified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants may be added to a composition comprising a NE and animmunogen, or, the adjuvant may be formulated with carriers, for exampleliposomes, or metallic salts (e.g., aluminium salts (e.g., aluminiumhydroxide)) prior to combining with or co-administration with acomposition comprising a NE and an immunogen.

In some embodiments, a composition comprising a NE and an immunogencomprises a single adjuvant. In other embodiments, a compositioncomprising a NE and an immunogen comprises two or more adjuvants (See,e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565;WO 99/11241; and WO 94/00153, each of which is hereby incorporated byreference in its entirety).

In some embodiments, a composition comprising a NE and an immunogen ofthe present invention comprises one or more mucoadhesives (See, e.g.,U.S. Pat. App. No. 20050281843, hereby incorporated by reference in itsentirety). The present invention is not limited by the type ofmucoadhesive utilized. Indeed, a variety of mucoadhesives arecontemplated to be useful in the present invention including, but notlimited to, cross-linked derivatives of poly(acrylic acid) (e.g.,carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides (e.g., alginate and chitosan), hydroxypropylmethylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose.Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, use of amucoadhesive (e.g., in a composition comprising a NE and immunogen)enhances induction of an immune response in a subject (e.g.,administered a composition of the present invention) due to an increasein duration and/or amount of exposure to an immunogen that a subjectexperiences when a mucoadhesive is used compared to the duration and/oramount of exposure to an immunogen in the absence of using themucoadhesive.

In some embodiments, a composition of the present invention may comprisesterile aqueous preparations. Acceptable vehicles and solvents include,but are not limited to, water, Ringer's solution, phosphate bufferedsaline and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed mineral or non-mineral oil maybe employed including synthetic mono-ordi-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.Carrier formulations suitable for mucosal, subcutaneous, intramuscular,intraperitoneal, intravenous, or administration via other routes may befound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa.

A composition comprising a NE and an immunogen of the present inventioncan be used therapeutically (e.g., to enhance an immune response) or asa prophylactic (e.g., for immunization (e.g., to prevent signs orsymptoms of disease)). A composition comprising a NE and an immunogen ofthe present invention can be administered to a subject via a number ofdifferent delivery routes and methods.

For example, an immunogenic compositions of the invention can beadministered to a subject (e.g., mucosally (e.g., nasal mucosa, vaginalmucosa, etc.)) by multiple methods, including, but not limited to: beingsuspended in a solution and applied to a surface; being suspended in asolution and sprayed onto a surface using a spray applicator; beingmixed with a mucoadhesive and applied (e.g., sprayed or wiped) onto asurface (e.g., mucosal surface); being placed on or impregnated onto anasal and/or vaginal applicator and applied; being applied by acontrolled-release mechanism; being applied as a liposome; or beingapplied on a polymer.

In some preferred embodiments, immunogenic compositions described hereinare administered mucosally (e.g., using standard techniques; See, e.g.,Remington: The Science and Practice of Pharmacy, Mack PublishingCompany, Easton, Pa., 19th edition, 1995 (e.g., for mucosal deliverytechniques, including intranasal, pulmonary, vaginal and rectaltechniques), as well as European Publication No. 517,565 and Illum etal., J. Controlled Rel., 1994, 29:133-141 (e.g., for techniques ofintranasal administration), each of which is hereby incorporated byreference in its entirety). Alternatively, immunogenic compositionsdescribed herein are administered dermally or transdermally, usingstandard techniques (See, e.g., Remington: The Science and Practice ofPharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995). Thepresent invention is not limited by the route of administration.

Mucosal vaccination, such as intranasal vaccination, may induce mucosalimmunity not only in the nasal mucosa, but also in distant mucosal sitessuch as the genital mucosa (See, e.g., Mestecky, Journal of ClinicalImmunology, 7:265-276, 1987). In addition to inducing mucosal immuneresponses, mucosal vaccination also induces systemic immunity (See,e.g., Example 10). In some embodiments, non-parenteral administration(e.g., mucosal administration of vaccines) provides an efficient andconvenient way to boost systemic immunity (e.g., induced by parenteralor mucosal vaccination (e.g., in cases where multiple boosts are used tosustain a vigorous systemic immunity)).

In some embodiments, a composition comprising a NE and an immunogen ofthe present invention may be used to protect or treat a subjectsusceptible to, or suffering from, disease by means of administering acomposition of the present invention via a mucosal route (e.g., anoral/alimentary or nasal route). Alternative mucosal routes includeintravaginal and intra-rectal routes. In preferred embodiments of thepresent invention, a nasal route of administration is used, termed“intranasal administration” or “intranasal vaccination” herein. Methodsof intranasal vaccination are well known in the art, including theadministration of a droplet or spray form of the vaccine into thenasopharynx of a subject to be immunized. In some embodiments, anebulized or aerosolized composition comprising a NE and immunogen isprovided. Enteric formulations such as gastro resistant capsules fororal administration, suppositories for rectal or vaginal administrationalso form part of this invention. Immunogenic compositions describedherein may also be administered via the oral route. Under thesecircumstances, a composition comprising a NE and an immunogen maycomprise a pharmaceutically acceptable excipient and/or include alkalinebuffers, or enteric capsules. Formulations for nasal delivery mayinclude those with dextran or cyclodextran and saponin as an adjuvant.

Immunogenic compositions described herein may also be administered via avaginal route. In such cases, a composition comprising a NE and animmunogen may comprise pharmaceutically acceptable excipients and/oremulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers ofvaginal creams and suppositories. In some embodiments, compositions ofthe present invention are administered via a rectal route. In suchcases, a composition comprising a NE and an immunogen may compriseexcipients and/or waxes and polymers known in the art for forming rectalsuppositories.

In some embodiments, the same route of administration (e.g., mucosaladministration) is chosen for both a priming and boosting vaccination.In some embodiments, multiple routes of administration are utilized(e.g., at the same time, or, alternatively, sequentially) in order tostimulate an immune response (e.g., using an immunogenic compositionsdescribed herein).

For example, in some embodiments, a composition comprising a NE and animmunogen is administered to a mucosal surface of a subject in either apriming or boosting vaccination regime. Alternatively, in someembodiments, a composition comprising a NE and an immunogen isadministered systemically in either a priming or boosting vaccinationregime. In some embodiments, a composition comprising a NE and animmunogen is administered to a subject in a priming vaccination regimenvia mucosal administration and a boosting regimen via systemicadministration. In some embodiments, a composition comprising a NE andan immunogen is administered to a subject in a priming vaccinationregimen via systemic administration and a boosting regimen via mucosaladministration. Examples of systemic routes of administration include,but are not limited to, a parenteral, intramuscular, intradermal,transdermal, subcutaneous, intraperitoneal or intravenousadministration. A composition comprising a NE and an immunogen may beused for both prophylactic and therapeutic purposes.

In some embodiments, immunogenic compositions described herein areadministered by pulmonary delivery. For example, immunogeniccompositions described herein can be delivered to the lungs of a subject(e.g., a human) via inhalation (e.g., thereby traversing across the lungepithelial lining to the blood stream (See, e.g., Adjei, et al.Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J.Pharmaceutics 1990; 63:135-144; Braquet, et al. J. CardiovascularPharmacology 1989 143-146; Hubbard, et al. (1989) Annals of InternalMedicine, Vol. III, pp. 206-212; Smith, et al. J. Clin. Invest. 1989;84:1145-1146; Oswein, et al. “Aerosolization of Proteins”, 1990;Proceedings of Symposium on Respiratory Drug Delivery II Keystone,Colorado; Debs, et al. J. Immunol. 1988; 140:3482-3488; and U.S. Pat.No. 5,284,656 to Platz, et al, each of which are hereby incorporated byreference in its entirety). A method and composition for pulmonarydelivery of drugs for systemic effect is described in U.S. Pat. No.5,451,569 to Wong, et al., hereby incorporated by reference; See alsoU.S. Pat. No. 6,651,655 to Licalsi et al., hereby incorporated byreference in its entirety)). Further contemplated for use in thepractice of delivery of immunogenic compositions described herein arethe wide range of mechanical devices designed for pulmonary and/or nasalmucosal delivery of pharmaceutical agents described herein

Thus, in some embodiments, an immunogenic composition described hereinmay be used to protect and/or treat a subject susceptible to, orsuffering from, a disease by means of administering a compositionscomprising a NE and an immunogen by mucosal, intramuscular,intraperitoneal, intradermal, transdermal, pulmonary, intravenous,subcutaneous or other route of administration described herein underconditions that induce an immunogen-specific immune response in thesubject. Methods of systemic administration of the vaccine preparationsmay include conventional syringes and needles, or devices designed forballistic delivery of solid vaccines (See, e.g., WO 99/27961, herebyincorporated by reference), or needleless pressure liquid jet device(See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No. 5,993,412, each ofwhich are hereby incorporated by reference), or transdermal patches(See, e.g., WO 97/48440; WO 98/28037, each of which are herebyincorporated by reference). The present invention may also be used toenhance the immunogenicity of antigens applied to the skin (transdermalor transcutaneous delivery, See, e.g., WO 98/20734; WO 98/28037, each ofwhich are hereby incorporated by reference). Thus, in some embodiments,the present invention provides a delivery device for systemicadministration, pre-filled with the vaccine composition of the presentinvention.

The present invention is not limited by the type of subject administered(e.g., in order to stimulate an immune response (e.g., in order togenerate protective immunity (e.g., mucosal and/or systemic immunity)))a composition of the present invention. Indeed, a wide variety ofsubjects are contemplated to be benefited from administration of acomposition of the present invention. In preferred embodiments, thesubject is a human. In some embodiments, human subjects are of any age(e.g., adults, children, infants, etc.) that have been or are likely tobecome exposed to a microorganism (e.g., bacteria of the genusBurkholderia). In some embodiments, the human subjects are subjects thatare more likely to suffer from opportunistic infection (e.g., a subjectwith CF or an immune suppressed subject (e.g., a subject with humanimmunodeficiency virus)). In some embodiments, the subjects arenon-human mammals (e.g., pigs, cattle, goats, horses, sheep, or otherlivestock; or mice, rats, rabbits or other animal). In some embodiments,compositions and methods of the present invention are utilized inresearch settings (e.g., with research animals). In some embodiments,the present invention provides a method to elicit an immune response(e.g., protective immune response) in infants (e.g., from about 0-2years old) by administering to the infant a safe and effective amount ofan immunogenic composition of the invention (e.g., a pediatric vaccine).Further embodiments of the invention include the provision of theimmunogenic Burkholderia nanoemulsion compositions of the invention foruse in medicine and the use of the Burkholderia nanoemulsioncompositions of the invention in the manufacture of a medicament for theprevention (or treatment) of disease caused by Burkholderia. In yetanother embodiment, the present invention is provides a method to elicitan immune response (e.g., a protective immune response) in the elderlypopulation (e.g., in a subject 50 years or over in age, typically over55 years and more generally over 60 years) by administering a safe andeffective amount of an immunogenic composition of the invention.

Immunogenic compositions described herein may be formulated foradministration by any route, such as mucosal, oral, topical, parenteralor other route described herein. The compositions may be in any one ormore different forms including, but not limited to, tablets, capsules,powders, granules, lozenges, foams, creams or liquid preparations.

Topical formulations may be presented as, for instance, ointments,creams or lotions, foams, and aerosols, and may contain appropriateconventional additives such as preservatives, solvents (e.g., to assistpenetration), and emollients in ointments and creams.

Topical formulations may also include agents that enhance penetration ofthe active ingredients through the skin. Exemplary agents include abinary combination of N-(hydroxyethyl) pyrrolidone and a cell-envelopedisordering compound, a sugar ester in combination with a sulfoxide orphosphine oxide, and sucrose monooleate, decyl methyl sulfoxide, andalcohol.

Other exemplary materials that increase skin penetration includesurfactants or wetting agents including, but not limited to,polyoxyethylene sorbitan mono-oleoate (Polysorbate 80); sorbitanmono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol polymer (TritonWR-1330); polyoxyethylene sorbitan tri-oleate (Tween 85); dioctyl sodiumsulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and otherpharmaceutically acceptable surfactants.

In certain embodiments of the invention, compositions may furthercomprise one or more alcohols, zinc-containing compounds, emollients,humectants, thickening and/or gelling agents, neutralizing agents, andsurfactants. Water used in the formulations is preferably deionizedwater having a neutral pH. Additional additives in the topicalformulations include, but are not limited to, silicone fluids, dyes,fragrances, pH adjusters, and vitamins.

Topical formulations may also contain compatible conventional carriers,such as cream or ointment bases and ethanol or oleyl alcohol forlotions. Such carriers may be present as from about 1% up to about 98%of the formulation. The ointment base can comprise one or more ofpetrolatum, mineral oil, ceresin, lanolin alcohol, panthenol, glycerin,bisabolol, cocoa butter and the like.

Immunogenic compositions described herein may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipuritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, preferablydo not unduly interfere with the biological activities of theimmunogenic compositions described herein. The formulations can besterilized and, if desired, mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like) that do not deleteriouslyinteract with the NE and immunogen of the formulation. In someembodiments, immunogenic compositions described herein are administeredin the form of a pharmaceutically acceptable salt. When used the saltsshould be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: hydrochloric, hydrobromic,sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulphonic, tartaric, citric, methane sulphonic, formic, malonic,succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include, but are not limited to, acetic acidand a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid anda salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).Suitable preservatives may include benzalkonium chloride (0.003-0.03%w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) andthimerosal (0.004-0.02% w/v).

In some embodiments, a composition comprising a NE and an immunogen isco-administered with one or more antibiotics. For example, one or moreantibiotics may be administered with, before and/or after administrationof a composition comprising a NE and an immunogen. The present inventionis not limited by the type of antibiotic co-administered. Indeed, avariety of antibiotics may be co-administered including, but not limitedto,

lactam antibiotics, penicillins (such as natural penicillins,aminopenicillins, penicillinase-resistant penicillins, carboxypenicillins, ureido penicillins), cephalosporins (first generation,second generation, and third generation cephalosporins), and other

actams (such as imipenem, monobactams,), β-lactamase inhibitors,vancomycin, aminoglycosides and spectinomycin, tetracyclines,chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin,metronidazole, polymyxins, doxycycline, quinolones (e.g.,ciprofloxacin), sulfonamides, trimethoprim, and quinolines.

There are an enormous amount of antimicrobial agents currently availablefor use in treating bacterial, fungal and viral infections. For acomprehensive treatise on the general classes of such drugs and theirmechanisms of action, the skilled artisan is referred to Goodman &Gilman's “The Pharmacological Basis of Therapeutics” Eds. Hardman etal., 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996,(herein incorporated by reference in its entirety). Generally, theseagents include agents that inhibit cell wall synthesis (e.g.,penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); andthe imidazole antifungal agents (e.g., miconazole, ketoconazole andclotrimazole); agents that act directly to disrupt the cell membrane ofthe microorganism (e.g., detergents such as polmyxin and colistimethateand the antifungals nystatin and amphotericin B); agents that affect theribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol,the tetracyclines, erythromycin and clindamycin); agents that alterprotein synthesis and lead to cell death (e.g., aminoglycosides); agentsthat affect nucleic acid metabolism (e.g., the rifamycins and thequinolones); the antimetabolites (e.g., trimethoprim and sulfonamides);and the nucleic acid analogues such as zidovudine, gangcyclovir,vidarabine, and acyclovir which act to inhibit viral enzymes essentialfor DNA synthesis. Various combinations of antimicrobials may beemployed.

The present invention also includes methods involving co-administrationof a composition comprising a NE and an immunogen with one or moreadditional active and/or immunostimulatory agents (e.g., a compositioncomprising a NE and a different immunogen, an antibiotic, anti-oxidant,etc.). Indeed, it is a further aspect of this invention to providemethods for enhancing prior art immunostimulatory methods (e.g.,immunization methods) and/or pharmaceutical compositions byco-administering a composition of the present invention. Inco-administration procedures, the agents may be administeredconcurrently or sequentially. In one embodiment, the compositionsdescribed herein are administered prior to the other active agent(s).The pharmaceutical formulations and modes of administration may be anyof those described herein. In addition, the two or more co-administeredagents may each be administered using different modes (e.g., routes) ordifferent formulations. The additional agents to be co-administered(e.g., antibiotics, adjuvants, etc.) can be any of the well-known agentsin the art, including, but not limited to, those that are currently inclinical use.

In some embodiments, a composition comprising a NE and immunogen isadministered to a subject via more than one route. For example, asubject that would benefit from having a protective immune response(e.g., immunity) towards a pathogenic microorganism may benefit fromreceiving mucosal administration (e.g., nasal administration or othermucosal routes described herein) and, additionally, receiving one ormore other routes of administration (e.g., parenteral or pulmonaryadministration (e.g., via a nebulizer, inhaler, or other methodsdescribed herein). In some preferred embodiments, administration viamucosal route is sufficient to induce both mucosal as well as systemicimmunity towards an immunogen or organism from which the immunogen isderived. In other embodiments, administration via multiple routes servesto provide both mucosal and systemic immunity. Thus, although anunderstanding of the mechanism is not necessary to practice the presentinvention and the present invention is not limited to any particularmechanism of action, in some embodiments, it is contemplated that asubject administered a composition of the present invention via multipleroutes of administration (e.g., immunization (e.g., mucosal as well asairway or parenteral administration of a composition comprising a NE andimmunogen of the present invention) may have a stronger immune responseto an immunogen than a subject administered a composition via just oneroute.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions, increasing convenience to thesubject and a physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer based systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109,hereby incorporated by reference. Delivery systems also includenon-polymer systems that are: lipids including sterols such ascholesterol, cholesterol esters and fatty acids or neutral fats such asmono-di- and tri-glycerides; hydrogel release systems; sylastic systems;peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5,736,152, each of which is hereby incorporated by reference and (b)diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,854,480, 5,133,974 and 5,407,686, each of which is hereby incorporatedby reference. In addition, pump-based hardware delivery systems can beused, some of which are adapted for implantation.

In preferred embodiments, a composition comprising a NE and an immunogenof the present invention comprises a suitable amount of the immunogen toinduce an immune response in a subject when administered to the subject.In preferred embodiments, the immune response is sufficient to providethe subject protection (e.g., immune protection) against a subsequentexposure to the immunogen or the microorganism (e.g., bacteria of thegenus Burkholderia) from which the immunogen was derived. The presentinvention is not limited by the amount of immunogen used. In somepreferred embodiments, the amount of immunogen (e.g., Burkholderiabacteria (e.g., Burkholderia cepacia) or one or more component partsthereof) in a composition comprising a NE and immunogen (e.g., for useas an immunization dose) is selected as that amount which induces animmunoprotective response without significant, adverse side effects. Theamount will vary depending upon which specific immunogen or combinationthereof is/are employed, and can vary from subject to subject, dependingon a number of factors including, but not limited to, the species, ageand general condition (e.g., health) of the subject, and the mode ofadministration.

In some embodiments, each dose (e.g., of a composition comprising a NEand an immunogen (e.g., administered to a subject to induce an immuneresponse (e.g., a protective immune response (e.g., protectiveimmunity))) comprises about 0.05-5000 μg of immunogen (e.g., recombinantand/or purified Burkholderia OMP protein). In some embodiments, eachdose (e.g., of a composition comprising a NE and an immunogen (e.g.,administered to a subject to induce an immune response (e.g., aprotective immune response (e.g., protective immunity))) comprises about0.05-5000 μg of the immunogen (e.g., recombinant and/or purifiedBurkholderia OMP protein) and comprises about 0.05-5000 μg of anotherimmunogen (e.g., recombinant and/or purified protein, adjuvant (e.g.,cholera toxin), etc.). In some embodiments, each dose will comprise1-500 μg, in some embodiments, each dose will comprise 350-750 μg, insome embodiments, each dose will comprise 50-200 μg, in someembodiments, each dose will comprise 25-75 μg of immunogen (e.g.,recombinant and/or purified protein (e.g., Burkholderia antigen). Insome embodiments, each dose comprises an amount of the immunogensufficient to generate an immune response. An effective amount of theimmunogen in a dose need not be quantified, as long as the amount ofimmunogen generates an immune response in a subject when administered tothe subject.

In some embodiments, it is expected that each dose (e.g., of acomposition comprising a NE and an immunogen (e.g., administered to asubject to induce and immune response)) is from 0.001 to 15% or more(e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more) by weightimmunogen (e.g., neutralized bacteria, or recombinant and/or purifiedprotein). In some embodiments, an initial or prime administration dosecontains more immunogen than a subsequent boost dose

In some embodiments, a composition comprising a NE and an immunogen ofthe present invention is formulated in a concentrated dose that can bediluted prior to administration to a subject. For example, dilutions ofa concentrated composition may be administered to a subject such thatthe subject receives any one or more of the specific dosages providedherein. In some embodiments, dilution of a concentrated composition maybe made such that a subject is administered (e.g., in a single dose) acomposition comprising 0.5-50% of the NE and immunogen present in theconcentrated composition. In some preferred embodiments, a subject isadministered in a single dose a composition comprising 1% of the NE andimmunogen present in the concentrated composition. Concentratedcompositions are contemplated to be useful in a setting in which largenumbers of subjects may be administered a composition of the presentinvention (e.g., an immunization clinic, hospital, school, etc.). Insome embodiments, a composition comprising a NE and an immunogen of thepresent invention (e.g., a concentrated composition) is stable at roomtemperature for more than 1 week, in some embodiments for more than 2weeks, in some embodiments for more than 3 weeks, in some embodimentsfor more than 4 weeks, in some embodiments for more than 5 weeks, and insome embodiments for more than 6 weeks.

Generally, the emulsion compositions of the invention will comprise atleast 0.001% to 100%, preferably 0.01 to 90%, of emulsion per ml ofliquid composition. It is envisioned that the formulations may compriseabout 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%,about 0.025%, about 0.05%, about 0.075%, about 0.1%, about 0.25%, about0.5%, about 1.0%, about 2.5%, about 5%, about 7.5%, about 10%, about12.5%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95% or about 98% of emulsion per ml ofliquid composition. It should be understood that a range between any twofigures listed above is specifically contemplated to be encompassedwithin the metes and bounds of the present invention. Some variation indosage will necessarily occur depending on the condition of the specificpathogen and the subject being immunized.

In some embodiments, following an initial administration of acomposition of the present invention (e.g., an initial vaccination), asubject may receive one or more boost administrations (e.g., around 2weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks,around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around4 months, around 6 months, around 9 months, around 1 year, around 2years, around 3 years, around 5 years, around 10 years) subsequent to afirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, and/or more than tenth administration. Although an understandingof the mechanism is not necessary to practice the present invention andthe present invention is not limited to any particular mechanism ofaction, in some embodiments, reintroduction of an immunogen in a boostdose enables vigorous systemic immunity in a subject. The boost can bewith the same formulation given for the primary immune response, or canbe with a different formulation that contains the immunogen. The dosageregimen will also, at least in part, be determined by the need of thesubject and be dependent on the judgment of a practitioner.

Dosage units may be proportionately increased or decreased based onseveral factors including, but not limited to, the weight, age, andhealth status of the subject. In addition, dosage units may be increasedor decreased for subsequent administrations (e.g., boostadministrations).

It is contemplated that the compositions and methods of the presentinvention will find use in various settings, including researchsettings. For example, compositions and methods of the present inventionalso find use in studies of the immune system (e.g., characterization ofadaptive immune responses (e.g., protective immune responses (e.g.,mucosal or systemic immunity))). Uses of the compositions and methodsprovided by the present invention encompass human and non-human subjectsand samples from those subjects, and also encompass researchapplications using these subjects. Compositions and methods of thepresent invention are also useful in studying and optimizingnanoemulsions, immunogens, and other components and for screening fornew components. Thus, it is not intended that the present invention belimited to any particular subject and/or application setting.

The formulations can be tested in vivo in a number of animal modelsdeveloped for the study of mucosal and other routes of delivery. As isreadily apparent, the compositions of the present invention are usefulfor preventing and/or treating a wide variety of diseases and infectionscaused by viruses, bacteria, parasites, and fungi, as well as foreliciting an immune response against a variety of antigens. Not only canthe compositions be used prophylactically or therapeutically, asdescribed above, the compositions can also be used in order to prepareantibodies, both polyclonal and monoclonal (e.g., for diagnosticpurposes), as well as for immunopurification of an antigen of interest.If polyclonal antibodies are desired, a selected mammal, (e.g., mouse,rabbit, goat, horse, etc.) can be immunized with the compositions of thepresent invention. The animal is usually boosted 2-6 weeks later withone or more—administrations of the antigen. Polyclonal antisera can thenbe obtained from the immunized animal and used according to knownprocedures (See, e.g., Jurgens et al., J. Chrom. 1985, 348:363-370).

In some embodiments, the present invention provides a kit comprising acomposition comprising a NE and an immunogen. In some embodiments, thekit further provides a device for administering the composition. Thepresent invention is not limited by the type of device included in thekit. In some embodiments, the device is configured for nasal applicationof the composition of the present invention (e.g., a nasal applicator(e.g., a syringe) or nasal inhaler or nasal mister). In someembodiments, a kit comprises a composition comprising a NE and animmunogen in a concentrated form (e.g., that can be diluted prior toadministration to a subject).

In some embodiments, all kit components are present within a singlecontainer (e.g., vial or tube). In some embodiments, each kit componentis located in a single container (e.g., vial or tube). In someembodiments, one or more kit component are located in a single container(e.g., vial or tube) with other components of the same kit being locatedin a separate container (e.g., vial or tube). In some embodiments, a kitcomprises a buffer. In some embodiments, the kit further comprisesinstructions for use.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 In Vivo Toxicity Studies

A nanoemulsion composition of the present invention was tested todetermine if pulmonary administration of the composition to a subjectwould elicit any histological changes and/or pathology.

A solution comprising 20% nanoemulsion (W₈₀5EC) and 0.1% EDTA wasadministered via a nebulizer. A custom murine nose only nebulizationchamber with PARI LC nebulizer (Midlothian, Va.) and compressor wasused. A 0.9% NaCl solution was used as a control. Using the nebulizer,mice were administered nebulized nanoemulsion+EDTA or NaCl solution for10 minutes. Twenty-four hours after administration, mice were sacrificedand physical properties and histology assessed.

Upon examination, there was an absence of histological changesindicating the absence of toxicity upon administration of nebulizednanoemulsion. Physical findings were normal.

Example 2 Killing Assays Utilizing Nanoemulsion Compositions andBacteria Found in the Respiratory Tract

It was determined whether a composition comprising nanoemulsion (W₈₀5EC)alone or in combination with EDTA and/or the presence of a hypertonicsalt solution would be able to attenuate growth and/or kill bacteriafound in the respiratory tract.

An overnight culture of Burkholderia cepacia or Pseudomonas aeruginosawas started in 6 ml of cation adjusted Mueller Hinton Broth (MHB) from afrozen bacterial stock at −80° C. The culture was incubated in a shakingincubator at 37° C. The following day, bacteria were brought tologarithmic growth phase by back diluting the overnight culture 1:4 withfresh MHB. Back diluted culture was incubated at 37° C. in the shakingincubator until the OD600 reached between 0.40 to 0.45. One ml of theculture was spun down at 3500 to 4000 rpm for 15 minutes. The bacterialpellet was resuspended in 1 ml of sterile 2×PBS, 12% or 14% saline.Appropriate dilutions were done with the same solutions to get desirednumbers of bacteria in 50 μl (OD600 of 0.5 approximately=10⁹ bacteria).Dilutions of the starting bacterial suspensions were plated onto LuriaBertani (LB) agar plates to estimate the starting bacterial counts.Double the concentrations of the desired final nanoemulsion andnanoemulsion with EDTA concentrations were made with sterile milliQwater. Fifty micro liters of the nanoemulsion was mixed with 50 μl ofthe bacteria and vortexed to mix the reaction. The mixture was incubatedat 37° C. for desired time intervals. Following incubation, the reactionwas diluted with 500 μl of 1×PBS and vortexed to mix the contents. Thecontents were centrifuged at 4000 rpm for 15 minutes to pellet thebacteria and separate the nanoemulsion. Supernatant containing thenanoemulsion was removed and the bacterial pellet resuspended in 1×PBS.Undiluted or dilutions of the resuspended bacterial pellet was platedonto LB agar plates for colony count. Log killing of the bacteria werecalculated from ratio with the starting numbers.

The following killing assays were performed:

Killing of Burkholderia cepacia in PBS by 20% Nanemulsion (NE) alone orwith 20 mM EDTA in 10 minutes (See FIG. 1);

Killing of B. cepacia in PBS by 20% NE alone or with 20 mM EDTA within20 minutes (See FIG. 2);

Killing of B. cepacia in PBS by 20% NE alone or with 20 mM EDTA within40 minutes (See FIG. 3);

Killing of B. cepacia in PBS by 20% NE alone or with 20 mM EDTA within40 or 60 minutes (See FIG. 4);

Killing of B. cepacia in hypertonic saline (6% NaCl) at 15 and 30minutes by NE alone and NE with EDTA (See FIG. 5);

Killing of B. cepacia and P. aeruginosa in 7% NaCl within 15 min (SeeFIG. 6); and

Killing of B. cepacia and P. aeruginosa (Mixed-culture) in 7% NaClwithin 15 minutes (See FIG. 7).

Nanoemulsion alone or in combination with EDTA (e.g., 10-20 mM EDTA) wasable to achieve complete killing of 10⁶ bacteria in PBS in 60 minutes.In the presence of hypertonic saline (e.g., 6-7% NaCl), the killingability of the nanoemulsion was strikingly enhanced, achieving completekilling within 15 minutes while in the presence of 20 mM EDTA. Also,nanoemulsions comprising a lower concentration of EDTA were able toachieve complete killing of bacteria in 30 minutes in the presence ofhypertonic saline. Thus, the present invention provides that ananoemulsion composition comprising EDTA can be used to kill (e.g.,completely) bacteria over a short time period (e.g., <60 minutes).Moreover, the present invention provides that nanoemulsion withhypertonic saline and EDTA can be used to kill bacteria over evenshorter time periods (e.g., <30 minutes, <15 minutes) and that theconcentration of EDTA and hypertonic saline solution alter the pace atwhich the nanoemulsion is able to eradicate the bacteria. The presentinvention also demonstrates that compositions of the present inventionare able to eradicate a mixed population of bacteria.

Example 3 Nanoemulsion Killing of Cystic Fibrosis (CF) Related BacteriaIncluding Multi-Drug Resistant Strains

Materials and Methods.

Bacterial strains and culture conditions. One hundred fifty isolateswere analyzed including 75 Burkholderia isolates and 75 isolatesbelonging to other CF-relevant species, including Pseudomonasaeruginosa, Achromobacter xylosoxidans, Stenotrophomonas maltophilia,Acinetobacter species, Pandoraea species (P. apista, P. pnomenusa, P.pulmonicola, P. norimburgensis, and P. sputorum), and Ralstonia species(R. mannitolilytica and R. pickettii). One hundred forty-five clinicalisolates were obtained from the Burkholderia cepacia Research Laboratoryand Repository (BcRLR, University of Michigan, Ann Arbor, Mich.). Thesewere recovered from 142 individuals between September 1997 and October2007 and were referred to the BcRLR from 62 CF treatment centers in theU.S. for analysis. The remaining five strains included environmentalisolates B. multivorans ATCC 17616 (American Type Culture Collection,Manassas, Va.), P. norimburgensis LMG 18379^(T) (BCCM/LMG BacteriaCollection, Laboratorium voor Micrbiologie Gent, Universiteit Gent,Ghent, Belgium) and B. pyrrocinia HI3642 (BcRLR collection), and theclinical type strains B. cenocepacia LMG 16656^(T) (aka J2315) and P.pulmonicola LMG 18106^(T). One hundred thirty four (91%) of the 147clinical isolates were recovered from persons with CF; 114 (78%) ofthese were from sputum culture, with the remainder from throat swab(n=17), endotracheal suction/tracheal aspiration (n=5), blood (n=5),bronchial lavage (n=3), and one each from maxillary sinus, peritonealcavity, and epiglottis. Forty nine (33%) of the 150 isolates weredefined as multi-drug resistant (resistant to all drugs tested in two ofthree antibiotic classes: lactams including carbapenems,aminoglycosides, and quinolones; See, e.g., Taccetti et al., Eur JEpidemiol. 1999 January; 15(1):85-8) based on susceptibility testingperformed at the referring microbiology laboratory; 20 (41%) of thesewere panresistant. Seventy two (48%) of the remaining isolates weresusceptible to at least one antibiotic, and susceptibility testingresults were unavailable for 29 (19%) isolates. All isolates wereidentified to the species level at the BcRLR by polyphasic analysesusing phenotypic and genotypic assays as described (See, e.g., Reik etal., J Clin Microbiol. 2005 June; 43(6):2926-8). The exception wasAcinetobacter, which was identified only to the genus level due to lackof definitive species-specific assays. All isolates were also subjectedto repetitive extragenic element-PCR (rep-PCR) typing using the BOX AIRprimer as previously described (See, e.g., Coenye et al., J ClinMicrobiol. 2002 September; 40(9):3300-7) to ensure that all 150 isolatesincluded in the test panel were genotypically distinct. Bacteria werestored at −80° C. in skim milk or Lauria-Bertani (LB) broth with 15%glycerol and recovered from frozen stock overnight at 37° C. onMueller-Hinton (MH) agar.

Nanoemulsion. Nanoemulsion P₄₀₇5EC, described herein, was manufacturedby NANOBIO Corp. (Ann Arbor, Mich.). P₄₀₇5EC droplets had a meanparticle diameter of 400 nm. Surfactants and food substances utilized inthe manufacture of P₄₀₇5EC were ‘Generally Recognized as Safe’ (GRAS) bythe FDA, and manufactured in accordance with Good ManufacturingPractices (GMP). The concentration of CPC was used as a surrogate forthe amount of P₄₀₇5EC used experimentally. P₄₀₇5EC was stable for noless than 12 months at 40° C.

Susceptibility testing. Because P₄₀₇5EC is opaque, the MICs of thiscompound for test bacteria were determined by using a modification ofthe Clinical and Laboratory Standards Institute (CLSI)-approvedmicrotiter serial dilution method (See, e.g., Clinical and LaboratoryStandards Institute. 2006. Approved standard M7-A7, seventh edition.Clinical and Laboratory Standards Institute, Wayne, Pa.). P₄₀₇5EC wasdiluted to a concentration of 2 mg/ml (of CPC) in MH broth supplementedwith 7% NaCl and 20 mM EDTA. Serial two-fold dilutions of thispreparation were made in unsupplemented MH broth and aliquoted into96-well flat bottom microtiter plates (100 μl/well). Bacteria fromovernight growth on MH agar were suspended in MH broth to a 0.5McFarland turbidity standard (absorbance of 0.08˜0.13 at 625 nm),further diluted 1:100 in MH broth, and added (5 μl/well) to the P₄₀₇5ECserial dilution wells. Appropriate controls, including wells withbacteria but no P₄₀₇5EC and wells with P₄₀₇5EC dilutions but nobacteria, were included on each plate. Microtiter plates were shakenbriefly, and 1 μl was removed from wells containing bacteria but noP₄₀₇5EC, diluted in 1 ml of MH broth, plated on MH agar (100 μl), andincubated for 24-48 h at 37° C. to determine bacterial concentrations ofinitial inoculums. Microtiter plates were then incubated at 37° C.without shaking To determine MBCs, 10 μl were removed from each wellafter overnight growth, spotted on MH agar, and incubated at 37° C.Colonies were enumerated 24 h later, and MBCs were recorded as theP₄₀₇5EC concentration with a 3 log decrease in CFU/ml compared to theinitial inoculum. To determine MICs, 10 μl of resazurin (R&D SYSTEMS,Minneapolis, Minn.) were added to each well and microtiter plates wereshaken briefly, covered with foil, and incubated at 37° C. withoutshaking Wells were visually inspected the next day, and the MIC wasrecorded as the lowest concentration of P₄₀₇5EC remaining a blue color.MIC results were further quantified by recording fluorescence on aspectrofluorometer at 560 nm excitation/590 nm emission.

Biofilm growth and susceptibility testing. To identify biofilm-formingisolates, bacteria were grown overnight in tryptic soy broth, adjustedto a 0.5 McFarland turbidity standard, and further diluted 1:10 in MHbroth, and 100 μl were seeded in triplicate in internal wells of 96-wellflat bottom microtiter plates. Negative control wells without bacteriawere included. To minimize evaporation, the remaining wells were filledwith MH broth, and the plate was wrapped with plastic wrap beforeincubating for 48 h at 37° C. without shaking Wells were gently washedtwice with phosphate buffered saline (PBS; pH 7.4), dried for 2 h at 37°C., and then stained with 1% crystal violet in water for 15 min. Stainedwells were washed 3 times with PBS, and crystal violet was solubilizedby the addition of absolute methanol. After incubation for 5 min at roomtemperature, the solubilized crystal violet was transferred to a newmicrotiter plate and scanned at 590 nm in a spectrophotometer. Abiofilm-forming isolate was defined as one where the average absorbanceof the 3 wells was greater than the average absorbance of the negativecontrol wells plus 3 standard deviations (See, e.g., Stepanovic et al.,J Microbiol Methods. 2000 April;40(2):175-9).

For biofilm susceptibility testing, isolates shown to produce biofilm bycrystal violet testing were grown in triplicate for 48 h as describedabove. Wells were gently washed twice with PBS before addition ofP₄₀₇5EC, serially diluted as described for planktonic MIC testing. Afterovernight incubation at 37° C., wells were again washed twice with PBS,and 100 μl of 10% resazurin in MH broth was added to wells. Plates werewrapped in plastic, covered with foil, and again incubated overnight at37° C. without shaking Plates were visually inspected the next day, andthe minimum biofilm inhibitory concentration (MBIC) was recorded as thelowest concentration of P₄₀₇5EC in which the wells were a blue color. Tocalculate minimum biofilm eradication concentrations (MBECs), biofilmwas resuspended in the same wells used for MBIC testing by shaking themicrotiter plate and then scraping the sides of the wells with a pipettip. Ten μl were removed from each well, spotted on MH agar plates andincubated at 37° C. After overnight growth, MBECs were recorded as thelowest P₄₀₇5EC concentration resulting in no growth (See, e.g., Tomlinet al., Can J Microbiol. 2001 October; 47(10):949-54). To confirminitial inoculum concentrations and to quantify viable bacteria inbiofilm grown cultures, colonies were enumerated from 10-fold serialdilutions of the 0.5 McFarland inoculating culture and from theuntreated positive control wells with resuspended biofilm for MBECcalculations.

Sputum preparation. Expectorated sputum, collected from CF patientsduring the course of routine care, was obtained from the University ofMichigan Health System clinical microbiology laboratory and stored at−80° C. Equal volumes of sputum from 15 individuals were pooled,mechanically sheared using a TISSUE MISER homogenizer (FISHERSCIENTIFIC, Pittsburgh, Pa.) at room temperature for 5 min at maximumspeed, and then incubated in an 80° C. water bath for 20 min. Processedsputum was divided into 10 ml aliquots, and 100 μl from each aliquot wasplated on MH agar and incubated for 48 h at 37° C. to confirm sterility.Aliquots were stored at −80° C.

Results.

Susceptibility of planktonic bacteria. Due to the opaque white color ofP₄₀₇5EC, the standard CLSI-approved microtiter serial dilution methodwas modified to include the addition of resazurin as an indicator ofbacterial viability. P₄₀₇5EC was tested in a concentration range of15.6-2000 μg/ml. MICs were defined as the lowest concentration ofP₄₀₇5EC that did not produce a color change from blue to pink (See FIG.8). Comparison of this visual inflection point with fluorometricanalysis showed that 63% of MIC wells had ≦1% of the metabolic activityof untreated control wells; 91% had ≦5% metabolic activity, and 96% had≦10% metabolic activity compared to control wells. The MIC results areshown in FIG. 11. All strains were inhibited by the concentrations ofP₄₀₇5EC tested. The MIC₅₀ for the entire panel of 150 strains was 31.2μg/ml; the MIC₉₀ was 125 μg/ml. Thirty eight strains (25%) wereinhibited by the lowest concentration of P₄₀₇5EC tested (15.6 μg/ml),and only a single strain each required a concentration of 250 μg/ml and500 μg/ml for inhibition. P₄₀₇5EC was slightly more active againstnon-Burkholderia strains (MIC₅₀ 31.2 μg/ml; MIC₉₀ 62.5 μg/ml) thanBurkholderia strains (MIC₅₀ 62.5 μg/ml; MIC₉₀ 125 μg/ml) (See FIG. 9).Activity was comparable across the 10 Burkholderia species tested. Nodifference was found in the activity of P₄₀₇5EC against multi-drugresistant Burkholderia strains compared to strains susceptible to one ormore antibiotics.

To evaluate the bactericidal activity of P₄₀₇5EC against planktonicbacteria, MBCs were determined on a subset of 34 strains including 22Burkholderia and 12 non-Burkholderia. All MBCs were within 1 dilution ofthe respective MICs for this subset of strains except for a single B.cenocepacia strain that had a MIC of 250 μg/ml and an MBC of 2000 μg/ml.A time-kill study of B. multivorans ATCC 17616 showed time- andconcentration-dependent killing, with a 99% decrease in bacterialviability within 90 min at a P₄₀₇5EC concentration two times greaterthan the MIC and complete killing within 30 min at concentrations eighttimes greater than the MIC (See FIG. 10A).

Susceptibility of bacteria grown in biofilm. To further assess theactivity of P₄₀₇5EC, bacteria grown as biofilms were tested forsusceptibility. Crystal violet staining identified 12 biofilm-formingstrains from among 25 strains screened from the test panel. Nine (75%)of the 12 strains showed decreased susceptibility to P₄₀₇5EC, defined asat least a four-fold increase in the MBIC compared to the MIC, whengrown as a biofilm (See FIG. 12). The median increase in MBIC comparedto the respective MIC of P₄₀₇5EC for the strains in this set waseight-fold. No evidence of tolerance of biofilm bacteria to P₄₀₇5EC wasobserved; the MBEC was the same as the respective MBIC for 10 of the 12strains.

Susceptibility of bacteria in CF sputum. The 12 biofilm-forming strainswere also tested for susceptibility to P₄₀₇5EC in the presence of CFsputum to model more closely the CF pulmonary microenvironment. The MBCsof P₄₀₇5EC for all 12 strains, grown under planktonic conditions,increased in the presence of 43% sputum (the highest sputumconcentration achievable in the microtiter assay) compared to therespective MBCs under standard conditions (See FIG. 12). Nevertheless,all strains remained susceptible to P₄₀₇5EC in the presence of sputum.CF sputum inhibited the activity of P₄₀₇5EC against planktonic bacteriain a concentration dependent manner (See FIG. 10B).

Example 4 Nanoemulsion Killing of Individual and Mixed BacterialBiofilms

A composition comprising nanoemulsion P₄₀₇5EC, 7% saline and 20 mM EDTAwas tested for its ability to kill bacteria present in biofilms(individual or mixed).

Biofilm Growth. Bacterial biofilms on polycarbonate membranes wereformed following the procedure described in Current Protocols inMicrobiology (John Wiley & Sons, Inc., NJ, USA). Polycarbonate membraneswere sterilized by exposing the membrane to UV light for 10 minutes eachside using sterile forceps or by steam sterilization at 121° C. for 20minutes. The sterile membrane was placed on the surface of an agarmedium with the shiny surface facing upwards. Overnight culture of thebacterial strain of interest was diluted with broth media to an OD₆₀₀ of0.05 and 0.5 μl of the diluted culture placed onto the shiny surface ofthe polycarbonate membrane on the agar plate medium. The plate with themembrane was incubated at 37° C. for 24 hours following which; themembrane is transferred to a new agar medium for another 24 hours forbiofilm formation.

Bactericidal Assay for Biofilm Bacteria. Polycarbonate membrane with thebiofilm was immersed in 250 μl of 20% P₄₀₇5EC with 20 mM EDTA and 7%sodium chloride (NaCl) in a 1.5 ml eppendorf tube for 3 hours at 37° C.Seven percent NaCl served as the negative control. Following incubation,the membrane was transferred to 15 ml sterile polypropylene tubescontaining 10 ml of sterile PBS. One ml of sterile PBS was added to theeppendorf reaction tubes, vortexed and spun at 3500 rpm for 15 minutes.Following centrifugation, the supernatant containing the NE was removedand the bacterial pellet resuspended in their respective 15 ml tube inwhich the polycarbonate membrane was collected. The 15 ml tube was thenvortexed at maximum speed for 3 minutes to get bacteria from membraneinto the PBS. One hundred microliters of undiluted and diluted treatedand control samples were plated onto LB agar plates for colony count.

As shown in FIGS. 13-16, 20% P₄₀₇5EC with 7% saline and 20 mM EDTA waseffective to kill bacteria present in biofilms comprising a singlebacterial strain (See, e.g., FIG. 13, biofilms comprising a plurality ofbacterial strains (See, e.g., FIGS. 14-15), as well as biofilmsgenerated from bacteria that escaped killing in a first round oftreatment (See, e.g., FIG. 16), attaining between a 4 to 9 log reductionin bacterial numbers.

Example 5

This example describes the effectiveness of various nanoemulsionformulations against various gram negative pathogens that may beassociated with CF, wounds, burns and burn patients, or otherenvironments.

As noted above, CF is characterized by chronic respiratory infectionthat begins early in life with Staphylococcus aureus and Haemophilusinfluenzae infections and later colonization with mucoid strains ofPseudomonas aeruginosa. In CF lung disease, early colonization withBurkholderia cepacia correlates with a poor prognosis in CF patients.Patients are often prescribed inhaled tobramycin to preventexacerbations of bacteria infections. With time, patients can becomeunresponsive to tobramycin therapy/prophylaxis. New inhaled topicalagents that are not cross-resistant to known antibiotics would be valuedas an alternative to inhaled tobramycin.

Several topical nanoemulsions were evaluated for microbiologicalactivity against gram-negative isolates, including P. aeruginosa and B.cepacia. All 3 nanoemulsions kill quickly in the presence of an outermembrane permeabilizer such as EDTA. The nanoemulsions were evaluatedagainst 23 of the 35 gram-negative isolates for cidal activity, and allwere bactericidal (85-95%). Thus, the one or more of these novelnanoemulsions can be used to prevent exacerbation of chronic pulmonaryinfections.

Methods:

MICs and MBCs were determined using CLSI standard methods. MICs tonanoemulsions were determined in the presence of 5 mM EDTA, a knownenhancer of nanoemulsion activity. The addition of alamar blue, a redeoxindicator that yields a colorimetric change in response to metabolicactivity, was used to determine the MICs of nanoemulsions that areopaque at higher concentrations.

Emulsion Manufacturing:

Nanoemulsions W₂₀5EC, P₄₀₇5EC, and W₂₀5GBA₂ED are oil-in-water emulsionsmanufactured from ingredients that are Generally Recognized As Safe(GRAS) with a cationic detergent (cetylpyridinium chloride (CPC) orbenzalkonium chloride (BA)) as active ingredients that have proven safefor oral human use. The emulsion is formed from highly purified oil,ethanol, a nonionic surfactant and water. The average nanoemulsiondroplet size was 180 nm for W₂₀5EC and 350 nm for P₄₀₇5EC andW₂₀5GBA₂ED, as measured by dynamic light scattering using a MalvernZetasizer Nano3600 (Malvern Instruments Ltd., Worcestershire, UK). Theformulations for W₂₀5EC and P₄₀₇5EC are described above. W₂₀5GBA₂ED (v/v%) is distilled water (18.93%), TWEEN 20 (5%), Glycerine (8%), Soybeanoil (64%), BTC 824 50% (4%) (Stepan, Northfield, Ill.), and EDTA(0.0745%). The formulation may be diluted in water (e.g., 60%W₂₀5GBA₂ED, 40% water) to provide 60% W₂₀5GBA₂ED. This material may bediluted to a 10% formulation with 10 mM EDTA (e.g., 76 g water per 24 g100 mM EDTA per 20 g 60% W₂₀5GBA₂ED). Thus, in some embodiments, aformulation comprising oil, water, glycerine, surfactant, and BTC(n-alkyl dimethyl benzyl ammonium chloride), and/or EDTA is provided.

Source of Isolates.

The source of the clinical isolates were blood stream or skin and softtissue isolates collected by JMI Laboratories in the past 2 years.

MIC and MBC Determinations.

MICs of W₂₀5EC±0, 5, 10, 15, and 20 mM EDTA were evaluated incation-adjusted Mueller-Hinton broth by microdilution per M7-A7 (2006).MICs for a panel of gram-negative isolates were determined for thenanoemulsions, W205EC, P4075EC, and W205GBA2ED in the presence of 5 mMEDTA. EDTA was used to permeabilize the gram-negative envelope, aidinginfusion of the nanodroplets to the cell membrane, resulting in lysis.Alamar blue was added to the assay panels 2 hours post-inoculation forenhanced MIC endpoint detection. MBC values were assessed for allnanoemulsions and a comparator compound (fusidicacid) by plating theentire broth content from the MIC well and from those four doublingdilutions above the MIC for each selected organism onto blood agargrowth media. Quantitative colony counts were performed on the initialinoculum. The lowest concentration of antimicrobial agent that killed≧99.9% of the starting test inoculum was defined as the MBC endpoint.See Tables 2, 3, and 4.

TABLE 2 MIC values of W205EC ± EDTA Bacterial Isolates 0 mM 5 mM 10 mM15 mM 20 mM K. pneumoniae 24-5A 64  8-16 8 8 8 E. coli ATCC 25922 16 1≦0.12 ≦0.12 ≦0.12 P. mirabilis 119-163A 128 ≦0.12* ≦0.12 ≦0.12 ≦0.12 A.baumannii 67-299A 32 ≦0.12* ≦0.12 ≦0.12 ≦0.12 P. aeruginosa ATCC >25632-64 32 32 32 27853 B. cepacia 30-492A >256 >256 >256 >256 >256 *EDTAalone inhibited these strains.

TABLE 3 Cidality of nanoemulsions against a subset (23) of gram-negativeisolates Compound (number of strains) MBC/MIC Levo- ratio W₂₀5EC P₄₀₇5ECW₂₀5GBA₂ED floxacin Cidal ↑ 1 7 10 9 9 2 6 7 7 8 4 4 2 2 1 Static ↓ 8 11 1 >8x 2 1 5 No growth* 3 3 3

TABLE 4 Escherichia coli (n = 5)

Klebsiella pneumoniae (n = 5) % % Antimicrobial susceptible/susceptible/ agent MIC₅₀ MIC₉₀ Range resistant

MIC₅₀ MIC₉₀ Range resistant W₂₀5EC 2 — 1-2 —/— 8 — 4-

—/— P₄₀₇5EC 4 — 4 —/— 8 —  8-16 —/— W₂₀5G BA₂ ED 2 — 2-4 —/— 4 — 4-8 —/—Ceftazidime <=1 — <=1->16 80.0/20.0 <=1 — <=1->16 80.0/20.0 Cefepime<=0.12 — <=0.12->16   80.0/20.0 <=0.12 — <=0.12->16   80.0/20.0Piperacillin/ 2 — 1-8 100.0/0.0  2 —  1-64 80.0/0.0  tazobactam Imipenem0.25 — <=0.12-0.5   100.0/0.0  0.25 — <=0.12-0.25  100.0/0.0  Gentamicin<=2 — <=2->8  80.0/20.0 <=2 — <=2 100.0/0.0  Tobramycin 0.5 — 0.25-16  80.0/20.0 0.25 — 0.25-16   80.0/20.0 Levofloxacin 0.03 — 0.03->8  60.0/40.0 0.06 — 0.06-8   80.0/20.0 Tetracycline >8 —  4->8 20.0/80.0<=2 — <=2-4  100.0/0.0  Colistin <=0.5 — <=0.5 —/— <=0.5 — <=0.5 —/—Proteus mirabilis (n = 5) Pseudomonas aeruginosa (n = 5) % %Antimicrobial susceptible/ susceptible/ agent MIC₅₀ MIC₉₀ Rangeresistant MIC₅₀ MIC₉₀ Range resistant W₂₀5EC 8 — 4-8 —/— 16 — 16-32 —/—P₄₀₇5EC 16 —  8-16 —/— 64 — 32-64 —/— W₂₀5G BA₂ ED 8 —  4-16 —/— 16 — 8-16 —/— Ceftazidime <=1 — <=1 100.0/0.0 2 — 2-4 100.0/0.0 Cefepime<=0.12 — <=0.12 100.0/0.0 4 — 1-8 100.0/0.0 Piperacillin/ <=0.5 —<=0.5-1    100.0/0.0 2 —  2-16 100.0/0.0 tazobactam Imipenem 1 —<=0.12-2    100.0/0.0 1 —  1->8  80.0/20.0 Gentamicin <=2 — <=2100.0/0.0 <=2 — <=2-4  100.0/0.0 Tobramycin 1 — 0.25-1   100.0/0.0 0.5 —0.25-1   100.0/0.0 Levofloxacin 0.12 — 0.06-2   100.0/0.0 1 — 0.25-4   80.0/0.0 Tetracycline >8 — >8   0.0/100.0 8 —  8->8   0.0/40.0Colistin >4 — >4 —/— 1 — <=0.5-2    100.0/0.0 Acinetobacter baumannii (n= 5)^(b) Stenotrophomonas maltophilia (n = 5)^(c) % % Antimicrobialsusceptible/ susceptible/ agent MIC₅₀ MIC₉₀ Range resistant MIC₅₀ MIC₉₀Range resistant W₂₀5EC <=0.12 — <=0.12-2 —/— <=0.12 — <=0.12 —/— P₄₀₇5EC<=0.12 — <=0.12-4 —/— <=0.12 — <=0.12 —/— W₂₀5G BA₂ ED <=0.12 — <=0.12-4—/— <=0.12 — <=0.12 —/— Ceftazidime 4 —    <=1->16 80.0/20.0 16 — 2->1640.0/40.0 Cefepime 2 —  <=0.12->16 80.0/20.0 >16 — 4->16 —/—Piperacillin/ 2 —   <=0.5->64 80.0/20.0 >64 — 8->64 —/— tazobactamImipenem <=0.12 —  <=0.12->8 80.0/20.0 >8 — 4->8  —/— Gentamicin <=2 —   <=2->8 80.0/20.0 >8 — <=2->8   —/— Tobramycin 0.5 —   0.25->1680.0/20.0 >16 — 0.5->16   —/— Levofloxacin 0.25 —   0.06->8 80.0/20.00.5 — 0.5-8    60.0/20.0 Tetracycline <=2 —    <=2->8 80.0/20.0 >8 —<=2->8   —/— Colistin <=0.5 —  <=0.5-2 100.0/0.0  <=0.5 — <=0.5-1    —/— Burkholderia cepacia (n = 5) % Antimicrobial susceptible/ agentMIC₅₀ MIC₉₀ Range resistant W₂₀5EC 256 —  64->256 —/— P₄₀₇5EC 256 —128->256 —/— W₂₀5G BA₂ ED 128 —  32->256 —/— Ceftazidime 2 — 2-4 100.0/0.0 Cefepime 8 — 4-16 —/— Piperacillin/ 4 — 2-32 —/— tazobactamImipenem 8 — 4-8  —/— Gentamicin >8 — >8 —/— Tobramycin >16 — >16  —/—Levofloxacin 2 — 1-2  100.0/0.0 Tetracycline >8 — >8 —/— Colistin — — >4—/—

indicates data missing or illegible when filed

Results:

All 3 nanoemulsions had activity against Gram-negative isolates (EDTAalone inhibited 3 and 5 strains of A. baumannii and S. maltophilia,respectively). This included isolates that were resistant to comparatoragents. MBCs were performed for 23 isolates; W₂₀5EC, P₄₀₇5EC, andW₂₀5GBA₂ED were bactericidal against 85, 95, and 90% of the isolates,respectively. No cross-resistance to any known antibiotic was observedfor any of the nanoemulsions.

Conclusions:

Nanoemulsions were broadly active against Gram-negative species,including multidrug-resistant isolates. The documented MICs were withinthe range of concentrations achievable with topical application to skinor mucosal tissues. One or more of the nanoemulsions may be useful forprophylaxis and/or therapeutic treatment of chronic pulmonary infectionsin cystic fibrosis patients.

Example 6

The purpose of this example was to determine if nanoemulsions accordingto the invention have activity against Haemophilus influenzae isolates.H. influenzae is an important respiratory pathogen implicated in acuteexacerbations of cystic fibrosis patients as well as upper and lowerrespiratory tract infections for non-CF normal individuals.

This example determined the minimum inhibitory concentration of threeexemplary nanoemulsions (W₂₀5EC, P₄₀₇5EC and W₂₀5GBA₂) plus comparatordrugs against clinical isolates of H. influenzae. The results, asdescribed in more detail below, show that P₄₀₇5EC and W₂₀5EC had similarMIC ranges of 1.88-3.75 μg/ml. W₂₀5GBA₂ had a MIC range of 8-16 μg/ml.The addition of EDTA at 1 mM to each well containing nanoemulsion had noeffect on the MIC. Concentrations of EDTA greater than 1 mM inhibitedthe growth of bacteria, likely reducing the concentration of cations tolevels below those that support growth. MBC data for the threenanoemulsions+1 mM EDTA were obtained for seven strains. The MBCs werewithin 4-fold of the respective MICs, consistent with each nanoemulsionhaving bactericidal activity against H. influenzae isolates.

A. Materials and Methods

Source of Drugs.

W₂₀5EC, lot x1151, at a concentration of 5000 μg/ml (stockconcentration) was a pooled sample of clinical trial material (NB-001),manufactured at Patheon, Mississauga, Ontario, Canada. P₄₀₇5EC, lotX1138, and W₂₀5GBA₂, lot X1103, were manufactured at NanoBioCorporation. Comparator compounds, azithromycin, ampicillin, cefepimeand clindamycin were purchased from The United States Pharmacopeia;catalog numbers were 1046056, 1033000, 1097636, 1136002, respectively.Tetracycline was purchased from Fluka Biochemika, catalog number 87128.The emulsions were produced by mixing a water immiscible oil phase withan aqueous phase. The base formulations are shown in Table 5 below andrepresent the neat emulsions, which were further diluted to the desired%. Compositions are w/w % unless otherwise noted.

TABLE 5 Nanoemulsions Nanoemulsion Component Weight Percent (w/w %)W₂₀5EC ED Distilled Water 23.418%  EDTA 0.0745%  CetylpyridiniumChloride 1.068% Tween 20  5.92% Ethanol  6.73% Soybean Oil 62.79%P₄₀₇5EC Distilled Water 23.49% CPC 1.068% Poloxamer 407  5.92% Ethanol 6.73% Soybean Oil, NP 62.79% W₂₀₅GBA₂ (v/v %) Distilled Water 20.93%BTC 824    2% Tween 20    5% Glycerine    8% Soybean Oil   64%

Source of Bacterial Strains.

Ten clinical isolates of H. influenzae were obtained from Case WesternReserve University, Cleveland, Ohio. The quality control isolate, ATCC49247, was obtained from the American Type Culture Collection.

Susceptibility Assays.

The susceptibility of H. influenzae isolates to five commercialantibiotics and three nanoemulsion formulations was evaluated using theguidelines published by Clinical Laboratory Standards Institute(Clinical and Laboratory Standards Institute. Performance Standards forAntimicrobial Susceptibility and Testing; Seventeenth InformationalSupplement. CLSI document MI00-517 (ISBN 1-56238-625-5). CLSI, 940 WestValley Road, Suite 1400, Wayne, Pa. 19087-1988, 2007). The ranges ofdrug concentrations tested were as follows: Azithromycin at 0.25-128μg/ml, Ampicillin at 0.063-32 μg/ml, Cefepime at 0.063-32 μg/ml,Clindamycin at 0.125-64 μg/ml, Tetracycline at 0.125-64 μg/ml, P₄₀₇5ECat 0.12-60 μg/ml cetylpyridinium chloride, W₂₀5EC at 0.12-60 μg/mlcetylpyridinium chloride, W₂₀5GBA₂ at 0.125-64 μg/ml benzalkoniumchloride. Since nanoemulsions are not a single component, the MICs/MBCsare expressed as the concentration of the cationic surfactant; W₂₀5ECand P₄₀₇5EC both contain cetylpyridinium chloride (CPC) as the cationicsurfactant, while W₂₀5GBA₂ contains benzalkonium chloride (BA) as thecationic surfactant. Isolates were also tested with the same range ofnanoemulsion concentrations+1-5 mM EDTA to see if the latter enhancedthe activity of the nanoemulsion. EDTA concentrations were tested aloneto ensure that chelation of cations by EDTA was sufficient to inhibitbacterial growth independent of the nanoemulsion.

Minimum inhibitory concentrations (MIC) were determined visually as thefirst totally clear well in the series of the 10 concentrations of eachdrug. For the nanoemulsions, because they are intrinsically opaque, theMIC was determined as the first well observed to have some clearing fromthe least concentrated to the most concentrated of the serial dilution.MBC values were assessed for the nanoemulsions+1 mM EDTA for 7 clinicalstrains and the ATCC 49257 isolate by plating 10 μl from the welldetermined to be the MIC and 4 wells above the MIC onto chocolate bloodagar media. The lowest concentration of antimicrobial agent that killed≧99.9% of the starting test inoculum was defined as the MBC. A compoundor nanoemulsion is defined as bactericidal if its MBC/MIC ratio was ≦4.

Results:

Data for the MICs and MBCs are shown in Tables 6-7. P₄₀₇5EC, W₂₀5EC, andW₂₀5GBA₂ were equally effective antimicrobials in the presence orabsence of 1 mM EDTA. The MIC₉₀ values for these compounds were 3.75,3.75, and 16 μg/ml, respectively (Table 6). By MIC₉₀ values, themajority of isolates were susceptible to azithromycin, cefepime,tetracycline and ampicillin, but were resistant to clindamycin andampicillin.

The MBC values were within 4-fold of the respective MICs for eachisolate tested (Table 7); thus each of the nanoemulsions werebactericidal for H. influenzae, with a range of MBCs for P₄₀₇5EC,W₂₀5EC, and W₂₀5GBA₂ of 1.88-7.5, 1.88-7.5 and 8-16 μg/ml, respectively.

Conclusions:

P₄₀₇5EC and W₂₀5EC appeared equally effective against clinical isolatesof H. influenzae. The benzalkonium chloride formulation, W₂₀5GBA₂, wastwo- to four-fold less active than the other nanoemulsions by MIC₉₀ andMBC values. EDTA at concentrations ≧2 mM EDTA inhibited the growth of H.influenzae under these growth conditions. The addition of EDTA was notnecessary to enhance the activity of any of the nanoemulsions.

TABLE 6 Susceptibility of H. influenzae clinical isolates tonanoemulsions and control antibiotics MIC (μg/ml) P₄₀₇5EC + W₂₀5EC +W₂₀5GBA₂ + 1 mM 1 mM 1 mM Strain P₄₀₇5EC^(a) EDTA W₂₀5EC^(a) EDTAW₂₀5GBA₂ ^(b) EDTA Azi^(c) Amp^(c) Cef^(c) Tet^(c) Cli^(c) 49247 1.881.88 1.88 0.94 8 4 1 4 1 16 8 30 1.88 0.94 1.88 0.94 4 8 2 0.25 0.1250.5 16 32 1.88 1.88 1.88 3.75 8 4 8 32 0.125 2 8 33 3.75 1.88 3.75 3.758 8 1 0.25 0.0625 1 16 34 3.75 3.75 3.75 3.75 8 8 1 >32 0.125 1 16 353.75 3.75 7.5  7.5  16 16 1 0.25 0.125 0.5 8 36 3.75 3.75 3.75 3.75 8 162 0.125 0.125 1 16 37 1.88 1.88 3.75 3.75 16 16 1 >32 0.125 1 4 38 3.753.75 3.75 3.75 16 16 1 0.25 0.0625 0.5 16 41 1.88 0.94 1.88 0.94 8 42 >32 0.25 1 8 42 1.88 1.88 1.88 3.75 8 8 1 >32 0.125 1 4 MIC 1.88-3.750.94-3.75 1.88-3.75 0.94-7.5 4-16 4-16 1-8 0.25->32 0.0625-1 0.5-16 4-16range MIC₉₀ 3.75 3.75 3.75 3.75 16 16 2 >32 0.125 2 16 ^(a)MIC valuereflects the amount of μg cetylpyridinium chloride/ml ^(b)MIC valuesreflects the amount of μg benzalkonium chloride/ml ^(c)Azi =azithromycin; Amp = ampicillin; Cef = cefepime; Tet = tetracycline; Cli= clindamycin

TABLE 7 Cidality of nanoemulsions against H. influenzae clinical isolateMBC (μg/ml) P407 5EC + W₂₀5EC + 1 mM W₂₀5GBA₂ + Strain 1 mM EDTA EDTA 1mM EDTA 49247 1.88 1.88 8 30 1.88 1.88 8 32 3.75 3.75 8 34 7.5 3.75 8 357.5 7.5 16 36 7.5 7.5 16 37 3.75 3.75 8 38 7.5 7.5 16

For Table 8 below, drug concentrations are in μg/ml for the comparators,as μg cetylpyridinium chloride (CPC)/ml for W₂₀5EC and P₄₀₇5EC and as μgbenzalkonium chloride (BA)/ml for W₂₀5GBA₂. NC is for Negative Controland GC is for Growth Control. The chart describes the wells of a 96 wellmicrotiter plate. The rows listed as I, J, K, and L are the rows of apartial plate used to evaluate the nanoemulsions with different EDTAconcentrations. For each clinical strain and control strain, the chartbelow was used for tracking the MIC and MBC data. If the wells were tooopaque to read the MIC( ), the MBC was relied upon to provide the MIC.In Table 8, rows with light highlighting contain plate counts for MBC.The second number is the plate count of a 10 μl sample from themicrotiter plate used to determine the MBC. The MBC was defined as≧3-log reduction of the inoculum. The darker highlighting corresponds toMIC.

TABLE 8 H. influenzae MIC of Nanoemulsions with EDTA Haemophilusinfluenzae MIC of Nanoemulsions with EDTA of Sep. 18, 2008

Azi = azithromycin; Amp = ampicillin; Cef = cefepine; Cli = clindamycin;Tet = tetracycline.

Example 7

The purpose of this study was to determine the MIC and MBC of fournanoemulsions (NEs) according to the invention and compare theperformance of the NEs to conventional antibiotics against MRSA. MRSA isone of the microorganisms that cause lung infections in young cysticfibrosis patients and is also implicated in infections of skin and softtissue, including burn wound infections. Because of its varied virulencemechanisms, infections caused by MRSA can be lethal.

The purpose of this example was to evaluate 4 nanoemulsions according tothe invention for use in the topical treatment of infectious diseases.The nanoemulsions tested were W₈₀5EC, W₂₀5ECEDL2, W₂₀ 5GBA₂ED andP₄₀₇5EC. This example determined the minimum inhibitory concentration(MIC) of the four nanoemulsions plus comparator drugs against 29clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).

The results shown below demonstrate that the MIC ranges for thenanoemulsions were 0.5-2 (W₈₀5EC), 0.5-4 (W₂₀5ECEDL2), 1-4 (W₂₀5GBA₂ED),1-4 μg/ml (P₄₀₇5EC), respectively. Minimum bactericidal concentration(MBC) data was also collected and the MBC ranges were 0.5-16 (W₈₀5EC),1->32 (W₂₀5ECEDL2), 2-16 (W₂₀5GBA₂ED), and 1->16 μg/ml (P₄₀₇5EC),respectively.

A. Methods and Materials

Source of Drugs and Isolates.

Twelve comparator compounds, clindamycin (USP #1136002), doxycycline(USP #1226003), erythromycin (Sigma #E0774), levofloxacin (Sigma#28266), mafenide acetate (USP #1373008), mupirocin (Sigma #M7694),silvadene (USP #61260), vancomycin (Sigma #V1764), Oxacillin (Sigma#28221), sulfamethoxazole (USP #1631001), trimethoprim (USP #1692505),and silver nitrate (VWR #VW3462-0) were compared to the fournanoemulsions. The four nanoemulsions were (a) W₈₀5EC, (b) W₂₀5ECEDL2,(c) W₂₀5GBA₂ED and (d) P₄₀₇5EC. See Table 10, which provides drug paneltemplates (with increasing dilutions across the panel). The emulsionswere produced by mixing a water immiscible oil phase with an aqueousphase. The W₂₀5ECEDL2 formulation was microfluidized. The baseformulations are shown in Table 9 below and represent the neatemulsions, which were further diluted to the desired %. Compositions arew/w % unless otherwise noted.

TABLE 9 Nanoemulsions Nanoemulsion Component Weight Percent (w/w %)W₈₀5EC Water 23.490%  Ethanol 6.730% Cetylpyridinium Chloride 1.068%Polysorbate 80 5.920% Refined Soybean Oil 62.790%  W₂₀5ECEDL2 DistilledWater 23.418%  EDTA 0.0745%  Cetylpyridinium Chloride 1.068% Tween 20 5.92% Ethanol  6.73% Soybean Oil 62.79% W₂₀5GBA₂ED Distilled Water20.93% (v/v %) EDTA 0.0745%  BTC 824    2% Tween 20    5% Glycerine   8% Soybean Oil   64% P₄₀₇5EC Distilled Water 23.49% CPC 1.068%Poloxamer 407  5.92% Ethanol  6.73% Soybean Oil, NP 62.79%

TABLE 10 Drug Panel Templates Drug Panel Templates Compound in μg/ml (%for AgNO₃) Compound 1 2 3 4 5 6 7 8 9 10 11 12 A Clindamycin 64 32 16 84 2 1 0.5 0.25 0.125 NC GC B Doxycycline 128 64 32 16 8 4 2 1 0.5 0.25NC GC C Erythromycin 128 64 32 16 8 4 2 1 0.5 0.25 NC GC D Levofloxacin64 32 16 8 4 2 1 0.5 0.25 0.125 NC GC E Mafenide acetate 128 64 32 16 84 2 1 0.5 0.25 NC GC F Mupirocin 64 32 16 8 4 2 1 0.5 0.25 0.125 NC GC GSilvadene 128 64 32 16 8 4 2 1 0.5 0.25 NC GC H Vancomycin 64 32 16 8 42 1 0.5 0.25 0.125 NC GC A Oxacillin 16 8 4 2 1 0.5 0.25 0.125 0.06250.03125 NC GC B Sulfamethazole 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 NC GCC Trimethoprim 64 32 16 8 4 2 1 0.5 0.25 0.125 NC GC D AgNO₃ 0.01 0.0050.0025 0.00125 0.00063 0.00031 0.00016 0.00008 0.00004 0.00002 NC GC EP₄₀₇5EC 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 NC GC F W₂₀5ECEDL2 32 16 8 42 1 0.5 0.25 0.125 0.0625 NC GC G W₂₀5GBA₂ED 64 32 16 8 4 2 1 0.5 0.250.125 NC GC H W₈₀5EC 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 NC GC

Source of Bacterial Strains.

Twenty-nine clinical isolates of MRSA were obtained from the Universityof Dentistry and Medicine of New Jersey. The quality control isolate,ATCC 29213, was obtained from the American Type Culture Collection.Table 11 below provides the phenotype/genotype of each strain.

TABLE 11 Phenotype and genotype of MRSA isolates Strain Phenotype orGenotype Number MLST spatype spa repeat pattern SCCmec PFGE PVL  2394ST59 17 Z1-D1-M1-D1-M1-N1-K1-B1 IV USA1000 +  2402  2926 ST5 2T1-J1-M1-B1-M1-D1-M1-G1-M1-K1 II −  9897 ST1 131 U1-I1-J1-F1-K1-B1-P1-E1IV USA400 + 11118 131 U1-I1-J1-F1-K1-B1-P1-E1 IV USA400 + 11512 ST36 16W1-G1-K1-A1-K1-A1-O1-M1-Q1-Q1-Q1 II − 11540 ST8 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV USA300 + 11554 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV + 13219 2 T1-J1-M1-B1-M1-D1-M1-G1-M1-K1IV − 13367 2 T1-J1-M1-B1-M1-D1-M1-G1-M1-K1 II − 13386 2T1-J1-M1-B1-M1-D1-M1-G1-M1-K1 II − 13408 2 T1-J1-M1-B1-M1-D1-M1-G1-M1-K1IV − 13606 2 T1-J1-M1-B1-M1-D1-M1-G1-M1-K1 II − 13610 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 I − 13643 1 Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1IV + 13693 7 Y1-H1-G1-C1-M1-B1-Q1-B1-L1-O1 IV − 13701 2T1-J1-M1-B1-M1-D1-M1-G1-M1-K1 II 13722 2 T1-J1-M1-B1-M1-D1-M1-G1-M1-K1II − 13756 2 T1-J1-M1-B1-M1-D1-M1-G1-M1-K1 II − 13759 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV − 13868 2 T1-J1-M1-B1-M1-D1-M1-G1-M1-K1II − 15337 18 W1-F1-K1-A1-O1-M1-Q1 II − 18998 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV USA300 + (MUP-R) 19001 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV USA300 + (MUP-R) 19017 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV USA300 + (MUP-R) 19024 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV USA300 + (MUP-R) 19047 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV + (MUP-R) 19069 ST8 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV USA300 + (MUP-R) 19156 1Y1-H1-G1-F1-M1-B1-Q1-B1-L1-O1 IV + (MUP-R)

Susceptibility Assays.

The susceptibility of MRSA to twelve traditional antimicrobials and fournanoemulsion formulations was tested using the guidelines published bythe Clinical Laboratory Standards Institute (Clinical and LaboratoryStandard Institute, “Antimicrobial Susceptibility and Testing;Seventeenth Informational Supplement. CLSI document M100-S17 (ISBN1-56238-625-5), CLSI, Wayne, Pa.). The ranges of drug concentrationswere as follows: clindamycin (64-0.125 μg/ml), doxycycline (128-0.25μg/ml), erythromycin (128-0.25 μg/ml), levofloxacin (64-0.125 μg/ml),mafenide acetate (128-0.25 μg/ml), mupirocin (64-0.125 μg/ml), silvadene(128-0.25 μg/ml), vancomycin (64-0.125 μg/m), oxacillin (16-0.03125μg/ml), sulfamethoxazole (32-0.0625 μg/ml), trimethoprim (64-125 μg/ml),silver nitrate (0.01-0.00002%), W₈₀5EC (32-0.0625 μg/ml), W₂₀5ECEDL2(32-0.0625 μg/ml), W₂₀5GBA₂ED (64-0.125 μg/ml), and P₄₀₇5EC (32-0.0625μg/ml).

Minimum inhibitory concentrations (MIC) were determined visually as thefirst totally clear well in the series of the 10 concentrations of eachdrug. For the nanoemulsions, because they are intrinsically opaque, theMIC was called as the first well observed to have some clearing from theleast concentrated to the most concentrated. Minimum bactericidalconcentrations (MBC) values were assessed for all the antimicrobialstested. The lowest concentration of antimicrobial agent that killed 2:99.9% of the starting test inoculum was defined as the MBC. A compoundor nanoemulsion is defined as bactericidal if its MBC/MIC ratio was ˜4.

B. Results

Data for the MICs and MBCs are shown in Tables 11 and 12. All fournanoemulsions were potent against the clinical isolates of MRSA, with nodifferential activity noted for community-acquired MRSA (SCCmec type IV)or hospital-associated MRSA (SCCmec types I-Ill). Further, thereappeared to be no cross-resistance of the nanoemulsions to any of theknown antibiotics. The MIC₉₀ values were 2 μg/ml for P₄₀₇5EC, W₂₀5ECEDL2and W₈₀5EC. W₂₀5GBA₂ED had a MIC₉₀ Of 4 μg/ml. The MIC₉₀ valuesindicated that this collection of MRSA was susceptible to oralantibiotics, doxycyline (MIC₉₀=4 μg/ml), sulfamethoxazole (MIC₉₀=4μg/ml), trimethoprim (MIC₉₀=0.5 μg/ml), and vancomycin (MIC₉₀=1 μg/ml).Isolates were generally resistant to oral antibiotics levofloxacin(MIC₉₀=32 μg/ml), clindamycin (MIC₉₀=>64 μg/ml) and erythromycin(MIC₉₀=>128 μg/ml). Topical antibiotics that are used in the treatmentof burn wound infections, silvadene and silver nitrate, had MIC₉₀ valuesof 32 μg/ml and 0.00063%, respectively. Mafenide acetate (Sulfamylon) isanother topical treatment for burn wound infections, but it wasuniformly inactive against MRSA (MIC₉₀=>128 μg/ml). Mupirocin is atopical treatment for skin and soft tissue infections and is also usedto eradicate carriage of MRSA; 31% of the isolates were resistant tothis antibiotic.

The MBC₉₀ values for all of the nanoemulsions were within four-fold ofthe respective MIC₉₀ values, indicating that the nanoemulsions arebactericidal to ˜90% of the isolates. Among the other 12 antibiotics,only vancomycin was bactericidal.

TABLE 12 Susceptibility of MRSA to nanoemulsions and comparatorcompounds: MIC analysis MIC (μg/ml) Rank Maf. Order Clind Dox Eryth LevoAcet Mup Silva Vanco Ox 29 >64 4 >128 >64 >128 >64 64 2 >16 28 >644 >128 >64 >128 >64 64 1 >16 27 >64 4 >128 32 >128 >64 32 1 >16 26 >644 >128 32 >128 >64 32 1 >16 25 >64 0.25 >128 32 >128 >64 32 1 >16 24 >640.25 >128 32 >128 >64 32 1 >16 23 >64 0.25 >128 32 >128 >64 32 1 >1622 >64 0.25 >128 16 >128 >64 32 1 >16 21 >64 0.25 >128 16 >128 16 321 >16 20 >64 0.25 >128 16 >128 0.25 32 1 >16 19 >64 0.25 >128 16 >1280.25 32 1 >16 18 >64 0.25 >128 8 >128 0.25 32 1 >16 17 >64 0.25 >1288 >128 0.25 32 1 >16 16 >64 0.25 128 8 >128 0.25 32 1 >16 15 >64 0.25 648 >128 0.25 32 1 >16 14 >64 0.25 64 8 >128 0.25 32 1 >16 13 >64 0.25 644 >128 0.25 32 1 >16 12 0.125 0.25 64 4 >128 0.25 32 1 >16 11 0.125 0.2564 4 >128 0.125 32 1 >16 10 0.125 0.25 64 4 >128 0.125 32 1 >16 9 0.1250.25 32 4 >128 0.125 32 0.5 >16 8 0.125 0.25 32 2 >128 0.125 32 0.5 >167 0.125 0.25 32 0.5 >128 0.125 32 0.5 >16 6 0.125 0.25 16 0.25 >1280.125 16 0.5 >16 5 0.125 0.25 8 0.25 >128 0.125 8 0.5 >16 4 0.125 0.250.25 0.25 >128 0.125 8 0.5 >16 3 0.125 0.25 0.25 0.125 >128 0.125 8 0.516 2 0.125 0.25 0.25 0.125 >128 0.125 8 0.5 16 1 0.125 0.25 0.25 0.125128 0.125 8 0.5 0.5 MIC₉₀ (15) >64 0.25 64 8 >128 0.25 32 1 >16 MIC₉₀(26) >64 4 >128 32 >128 >64 32 1 >16 MIC (μg/ml) Rank Silver W₂₀5ECW₂₀5G Order Sulfa Trim Nitrate P₄₀₇5EC ED L2 BA₂ ED W₈₀5EC 29 >32 >640.00250 4 4 4 2 28 >32 1 0.00125 4 2 4 2 27 4 1 0.00125 2 2 4 2 26 4 0.50.000626 2 2 4 2 25 2 0.25 0.000626 2 2 4 2 24 2 0.125 0.000626 2 2 4 223 2 0.125 0.000626 2 2 2 2 22 1 0.125 0.000626 2 2 2 1 21 1 0.1250.000626 2 2 2 1 20 1 0.125 0.000626 2 2 2 1 19 1 0.125 0.000312 2 1 2 118 1 0.125 0.000312 2 1 2 1 17 1 0.125 0.000312 1 1 2 1 16 1 0.1250.000312 1 1 2 1 15 1 0.125 0.000312 1 1 2 1 14 1 0.125 0.000312 1 1 2 113 1 0.125 0.000312 1 1 2 1 12 0.5 0.125 0.000312 1 1 2 1 11 0.5 0.1250.000312 1 1 2 1 10 0.5 0.125 0.000312 1 1 2 1 9 0.5 0.125 0.000312 1 12 1 8 0.5 0.125 0.000312 1 1 2 1 7 0.5 0.125 0.000312 1 1 2 1 6 0.250.125 0.000312 1 1 2 1 5 0.25 0.125 0.000312 1 1 2 1 4 0.25 0.1250.000312 1 1 2 1 3 0.25 0.125 0.000312 1 1 1 0.5 2 0.25 0.125 0.000312 10.5 1 0.5 1 0.0625 0.125 0.000312 1 0.5 1 0.5 MIC₉₀ 1 0.125 0.00031 1 12 1 (15) MIC₉₀ 4 0.5 0.00063 2 2 4 2 (26)

TABLE 13 Susceptibility of MRSA to nanoemulsions and comparatorcompounds: MBC analysis MBC (μg/ml) Maf. Rank Order Clind Dox Eryth LevoAcet. Mup Silva. Vanco Ox Sulfa 29 >64 >128 >128 >64 >128 >64 >1284 >16 >32 28 >64 >64 >128 >64 >128 >64 >128 4 >16 >3227 >64 >64 >128 >64 >128 >64 >128 4 >16 >3226 >64 >64 >128 >64 >128 >64 >128 2 >16 >3225 >64 >64 >128 >64 >128 >64 >128 2 >16 >3224 >64 >4 >128 >64 >128 >64 >128 2 >16 >32 23 >64 >4 >12864 >128 >64 >128 2 >16 >32 22 >64 >4 >128 64 >128 >64 >128 2 >16 >1621 >64 >4 >128 >32 >128 >64 >128 2 >16 >16 20 >64 >4 >12832 >128 >8 >128 2 >16 >16 19 >64 >4 >128 32 >128 >4 >128 1 >16 >1618 >64 >4 >128 32 >128 >4 >128 1 >16 >16 17 >64 >4 >128 32 >128 >4 >1281 >16 >16 16 >64 >4 >128 32 >128 >4 >128 1 >16 >16 15 >64 >4 >12816 >128 >4 >128 1 >16 >16 14 >64 >4 >128 16 >128 >2 128 1 >16 >1613 >64 >4 >128 16 >128 >2 128 1 >16 >16 12 >2 >4 >128 8 >128 >2 1281 >16 >8 11 >2 >4 >128 8 >128 >2 128 1 >16 >8 10 >2 >4 >128 8 >128 >2128 1 >16 >8 9 >2 >4 >128 8 >128 >2 128 1 >16 >8 8 >2 >4 >128 4 >128 >2128 1 >16 >8 7 >2 >4 >128 1 >128 >2 128 1 >16 >4 6 >2 >4 >1280.5 >128 >2 64 1 >16 >4 5 >2 >4 64 0.25 >128 >2 64 1 >16 >4 4 >2 >4 >40.25 >128 >2 64 1 >16 >4 3 >2 2 >4 0.25 >128 1 64 0.5 >16 >1 2 2 1 >40.25 >128 1 64 0.5 >16 1 1 1 1 >4 0.25 >128 1 64 0.5 1 0.0625 MBC₉₀(15) >64 >4 >128 16 >128 >2 128 1 >16 >16 MBC₉₀(26) >64 >64 >128 >64 >128 >64 >128 2 >16 >32 MBC (μg/ml) Silver W₂₀5ECW₂₀5G Rank Order Trim Nitrate P₄₀₇5EC ED L2 BA₂ ED W₈₀5EC 29 >640.01 >16 >32 16 16 28 >16 >0.01 16 8 8 4 27 >16 >0.01 16 8 8 426 >4 >0.01 8 8 8 4 25 >2 >0.005 8 8 8 4 24 >2 >0.005 8 4 8 423 >2 >0.005 4 4 4 4 22 4 0.005 4 4 4 4 21 2 0.0025 4 4 4 4 20 2 0.00254 4 4 4 19 2 0.0025 4 4 4 4 18 1 0.0025 4 4 4 2 17 1 0.0025 4 4 4 2 160.5 0.0025 4 4 4 2 15 0.5 0.0025 2 4 4 2 14 0.5 0.0025 2 2 4 2 13 0.50.0025 2 2 4 2 12 0.5 0.0025 2 2 4 2 11 0.25 0.0025 2 2 4 2 10 0.250.0025 2 2 4 2  9 0.25 0.00125 2 2 4 2  8 0.25 0.00125 2 2 4 2  7 0.250.00125 2 2 4 2  6 0.25 0.00125 2 2 4 2  5 0.125 0.00125 2 2 4 1  40.125 0.00125 2 2 4 1  3 0.125 0.00125 2 2 2 1  2 0.125 0.00125 1 1 2 1 1 0.0125 0.00125 1 1 2 0.5 MBC₉₀ (15) 0.5 0.0025 2 2 4 2 MBC₉₀(26) >4 >0.01 8 8 8 4

C. Conclusions

W₈₀5EC, W₂₀5ECEDL2, W₂₀5GBA₂ED and P₄₀₇5EC appeared equally effectiveagainst clinical isolates of MRSA, with MIC₉₀ values of 2 or 4 μg/ml. Inaddition, based on the ratio of MBC₉₀/MIC₉₀, the four nanoemulsions werebactericidal. As expected, the MRSA were multidrug-resistant,emphasizing the need for new agents for treating MRSA infections. Sincenanoemulsions according to the invention also have activity againstserious gram-negative pathogens, such as Pseudomonas aeruginosa andBurkholderia spp. (LiPuma et al., “In vitro activities of a novelnanoemulsion against Burkholderia and other multidrug-resistant cysticfibrosis-associated bacterial species,” Antimicrob. Agents Chemother.,53:249-255 (2009)), the nanoemulsions are useful for inhalationtreatment/maintenance of cystic fibrosis patients. In addition, thenanoemulsions are useful in treating burn wounds to prevent/treatinfections of the pathogenic agents described herein.

Example 8

The purpose of this example was to determine the minimum inhibitoryconcentration (MIC) and the minimum bactericidal concentration (MBC) ofan exemplary nanoemulsion (P₄₀₇5EC) side by side with comparator drugsagainst clinical isolates of Pseudomonas aeruginosa, Burkholderiacenocepacia, Acinetobater baumanni, Stenotrophomonas maltophilia frompatients suffering from cystic fibrosis (CF).

Summary of Results for MIC and MBC: P₄₀₇5EC+EDTA had an MIC range of<4-16, <4-64, 2-8, <1-16 μg CPC/ml, respectively. The addition of EDTAat 5 mM for the Pseudomonas and Burkholderia and 1 mM to Acinetobacterand Stenotrophomonas to each well containing nanoemulsion improved theMIC. MBC data for the nanoemulsion+EDTA were obtained for 20Pseudomonas, 10 Burkholderia, 10 Acinetobacter and 11 Stenotrophomonas.For three genera of bacteria tested, the data suggested bacteriostaticactivity. The MBCs were within 4-fold of the respective MICs for theStenotrophomonas, consistent with the nanoemulsion having bactericidalactivity.

Summary of Synergy Results: In addition to the standardized MIC and MBCdeterminations, checkerboard synergy studies were conducted to evaluatethe potential of P₄₀₇5EC+EDTA to synergize or antagonize traditionalantimicrobials commonly used to treat patients with cystic fibrosis. Thefractional inhibitory concentration (FIC) index and the fractionalbactericidal concentration (FBC) index were determined to judge if a twodrug combination was synergistic, antagonistic or indifferent to oneanother. Ten strains of Burkholderia, 10 strains of Stenotrophomonas and10 strains of Acinetobacter were tested to determine a shift in MIC whenP₄₀₇5EC+EDTA was in combination with either colistin or tobramycin, twotraditional antimicrobials used in the lungs of CF patients to treatchronic lung infections. P₄₀₇5EC+EDTA in combination with colistin wasfound to be synergistic for 90% (in terms of the FIC) and 70% (in termsof the FBC) of the Stenotrophomonas strains, but indifferent, only 20%synergy in by the FIC and 0% by the FBC, when in combination withtobramycin. For the Acinetobacter strains, P₄₀₇5EC+EDTA in combinationwith colistin was found to be indifferent, only 20% synergy in by theFIC and 0% by the FBC, as well as when in combination with tobramycin,only 10% synergy in by the FIC and 10% by the FBC. For the Burkholderiastrains, P₄₀₇5EC+EDTA in combination with colistin was found to beindifferent, only 30% synergy in by the FIC and 10% by the FBC, but whenin combination with tobramycin, 50% synergy in by the FIC and 20% by theFBC.

A. Materials and Methods

Multidrug-resistant Gram-negative bacteria isolated from CF patientswere tested in this example. MICs and MBCs were determined using CLSIguidelines and standard methods M7-A7 and M100-S17. Depending on thegenus, MICs were performed in the presence of 1 or 5 mM EDTA, apermeability enhancer of NE activity. Stenotrophomonas andAcintetobacter had 1 mMEDTA in the nanoemulsion. Pseudomonas andBurkholderia had 5 mM EDTA in the nanoemulsion. This was the case duringthe standard MIC and MBC determination as well as the Checkerboardsynergy work and time kill study to be described below. The addition ofalamar blue, a redox indicator that yields a colorimetric change inresponse to metabolic activity, was used to determine the MICs of the NE(P₄₀₇5EC) because of opacity at higher concentrations.

Checkerboard synergy studies were carried out by using the MIC and MBCdata collected, microtiter plates with a combination for P₄₀₇5EC+EDTAand colistin or P₄₀₇5EC+EDTA and tobramycin.

The Fractional Inhibitory Concentration (FIC) Index (see e.g., FIG. 19)examines the ratio of the MIC of a single drug when in combination withanother to the MIC of that drug alone. The reduction of the MIC when adrug is in combination results in a fraction that is less than one.

X=MIC of Drug in Combination/MIC of Drug along

This ratio is calculated for both drug A and drug B. The fractions areadded together. The summation is compared to the following ranges:

Synergism: Sum of FIC for the two drugs ≦0.5;

Indifference: Sum of FIC for the two drugs >0.5 to ≦4;

Antagonism: Sum of FIC for the two drugs >4.

In FIG. 19A, Row D denotes the previously determined MIC used to chosethe flanking concentrations of the two drugs being combined. In FIG.19B, the horizontal slash marks on the X and Y axis indicate typical MICconcentration of each drug alone.

The time-kill study was done over a brief time curve of 10, 20, 30minutes including a 0 minute non-treated control was conducted to fixsamples for pictures under the electron microscope.

B. Source of Isolates

46 isolates were evaluated, all of which were obtained from theUniversity of Maryland-Baltimore School of Dentistry. The isolatesreceived were as follows: 20 Pseudomonas, 11 Burkholderia, 10Acinetobacter and 5 Stenotrophomonas. The P. aeruginosa isolates haddefined lipid A modifications that have been documented in many CFpatients. 10 additional isolates of Stenotrophomonas were received froma second source (Eurofins, Va.). These 10 are the last on the list andare the isolates with the ages of the individuals the samples were takenfrom. The isolates are summarized in Table 14 below.

TABLE 14 Pseudomonas aeruginosa, Burkholderia cenocepacia, Acinetobacterbaumannii, Stenotrophomonas maltophilia isolates PseudomonasAminoarabinose modification Isolate Patient aeruginosa NanoBio numberProbable colistin resistance Location Identifier 1 NB1 − CF Patient A 2NB2 − CF Patient A 3 NB3 + CF Patient B 4 NB4 + CF Patient B 5 NB5 + CFPatient C 6 NB6 + CF Patient C 7 NB7 + CF Patient D 8 NB8 + CF Patient D9 NB9 − CF Patient E 10 NB10 − CF Patient E 11 NB11 − CF Patient F 12NB12 − CF Patient G 13 NB13 − CF Patient G 14 NB14 − CF Patient H 15NB15 − CF Patient I 16 NB16 + CF Patient J 17 NB17 − CF Patient K 18NB18 − CF Patient L 19 NB19 − CF Patient M 20 NB20 + CF Patient NBurkholderia Isolate Patient cenocepacia Nano Bio number GenomovarLocation Identifier 1 NB21 B. c. Genomovar I CF Patient A 2 NB22 B. c.Genomovar I CF Patient B 3 NB23 B. c. Genomovar III CF Patient C 4 NB24B. c. Genomovar III CF Patient D 5 NB25 B. c. Genomovar III CF Patient E6 NB26 B. c. Genomovar III CF Patient F 7 NB27 B. c. Genomovar III CFPatient G 8 NB28 B. c. Genomovar IV CF Patient H 9 NB29 B. c. GenomovarIV CF Patient I 10 NB30 B. c. Genomovar IV CF Patient J 11 NB28b B.vietnamienis Genomovar V Acinetobacter Isolate Patient baumannii NanoBio number Colistin Susceptibility Location Identifier 1 NB31 Sensitivewound, intraabdominal Unknown 2 NB32 Sensitive abcess Unknown 3 NB33Resistant peritoneal fluid Unknown 4 NB34 Sensitive sub-clav bloodUnknown 5 NB35 Resistant blood Unknown 6 NB36 Resistant wound Unknown 7NB37 Sensitive tissue Unknown 8 NB38 Sensitive urine Unknown 9 NB39Resistant CSF Unknown 10 NB40 Resistant urine Unknown Isolate PatientStenotrophomonas Nano Bio number Locution Identifier 1 NB41 CF Patient A2 NB42 CF Patient B 3 NB43 CF Patient C 4 NB44 CF Patient D 5 NB45 CFPatient E Date Stenotrophomonas Isolation Source maltophilia Short Id.Region Year Description Age 2482274 1 NEW ENGLAND 2006 Sputum 14 24822702 EAST NORTH CENTRAL 2007 Sputum 10 2482275 3 MOUNTAIN 2007 Sputum 172482269 4 MID ATLANTIC 2008 Sputum 15 2482273 5 SOUTH ATLANTIC 2008Sputum 17 2482276 6 EAST SOUTH CENTRAL 2008 Sputum 9 2482267 7 WESTNORTH CENTRAL 2008 Sputum 13 2482271 8 WEST SOUTH CENTRAL 2008 Sputum 42482272 9 PACIFIC 2008 Sputum 16 2482268 10 SOUTH ATLANTIC 2008 Sputum 1

C. Source of Drugs

The drugs and the source thereof used in the drug panel plates aresummarized in the table below.

TABLE 15 Drugs used in drug panel plates. Ingredient Manufacturer Lot #Cefepime USP H0G27S Colistin USP G-1 Imipenem USP H0E040 LevofloxacinSigma 1333515 Ceftazidime USP H Tobramycin USP L0E077 Ceftoxin USPJ0E038 Piperacillin USP H P407-5EC NanoBio XI138 and XI180

The composition of the P₄₀₇-5EC is provided in Table 16, below. Theemulsion was produced by mixing a water immiscible oil phase with anaqueous phase. The base formulation is below and represents the neatemulsion, which was further diluted to the desired %.

TABLE 16 Lot # A0499 Neat P₄₀₇ SEC Ingredient w/w % Sterile distilledwater 23.49 CPC 1.068 Poloxamer 407 5.92 Ethanol 6.73 Soybean oil 62.79

D. Preparation of Drug Concentrations

The preparation of drug concentration is described below in Tables 17and 18. Specifically, Table 17 provides details regarding thepreparation of the 96-well drug panel plates, and Table 18 providesdetails regarding the preparation of the 96-well checkerboard synergyplates.

TABLE 17 Details describing the preparation of the 96-well drug panelplates. Exp. Stock Molecular Potency Concentrations Volume RequiredWeight (ug/ Starting in Concentration Weight Name & Lot # g/mole mg) atX ug/mL: Solvent/Diluent mL (ug/mL) Needed (mg) Cefepime-USP 571.5 86532 Phos. Buffer 5.00 3200 18.50 Lot H0G278 pH 6 Colistin 1163 629 8water 10.00 800 12.72 (Polymyxin E)- USP Lot G-1 Imipenem-USP 317.36 92932 Phos. Buffer 5.00 3200 17.22 Lot H0E040 pH 7.2 Levofloxacin- 361.37999.00 16 ½ volume 10.00 1600 16.02 Sigma 1333515 water, then 0.1 mol/LNaOH dropwise to dissolve Piperacillin- 535.57 984 1024 ½ volume 2.00102400 208.13 USP Lot H water, then 0.1 mol/L NaOH dropwise to dissolveTobramycin- 467.52 970 32 water 5.00 3200 16.49 USP Lot L0E077Cefoxitin-USP 449.44 992 256 ½ volume 2.00 25600 51.61 Lot J0E038 water,then 0.1 mol/L NaOH dropwise to dissolve Ceftazidime-Lot H 535.57 852 32Calcium 2.00 3200 7.51 Carbonate Sln 10% of the weight of compound to bedissolved YELLOW HIGHLIGHT INDICATES POTENCY TAKEN FROM PERCENT PURITY(99% = 999 ug/mg). 1st Dilute ½, nine times from stock in solvent 2ndDilute 1/50 in medium in a respective well of a 12 well trough. 3rdTransfer 50 uL from each well of the reservoir into the respective wellfor each agent at each concentration. Required with or Solvent StockConcentration Volume Needed Volume without and Volume in (mM or ofInitial Stock Needed of Nanoemulsion EDTA Volume mL ug/mL) (mL) broth(mL) X1138 or with 1 mM Water 4.00 512 0.68 3.32 X1180 = P₄₀₇5EC (@ 6mg/mL) X1138 or with 5 mM Water 4.00 2056 2.74 1.26 X1180 = P₄₀₇5EC (@ 6mg/mL) ** Because of the high concentration of this test antimicrobial,a 100x stock could not be made. The calculations provided a 2x stockthat is diluted in broth from the start and through the nine ½dilutions. A 1/50 dilution at any time is not required because thedilutions are made directly in broth. X mM EDTA Diluted EDTA in BrothBroth Stock @ 100x volume Volume Total Volume EDTA Stock 100 800 2001000 solution starting 500 0 1000 1000 at 500 mM EDTA used to make thesedilutions. For the required concentration of EDTA, a 1/50 dilution ofthe EDTA 100x stock was made in each well of the 12 well troughcontaining nanoemulsion. Only 10 wells of the 12 well trough containdrug, the last two wells are for broth only which set up the negativeand positive growth controls.

TABLE 18 Details describing the preparation of the 96-well checkerboardsynergy plates. Required with or Solvent Stock Concentration VolumeNeeded Volume without and Volume (mM or of Initial Stock Needed ofNanoemulsion EDTA Volume in mL ug/mL) (mL) broth (mL) X1180 = P₄₀₇5ECwith 1 or Broth 4.00 Varied on plate (@ 6 mg/mL) 5 mM for each strain ofbacteria Colistin no Broth 4.00 Varied on plate (Polymyxin E)- for eachstrain USP Lot G-1 of bacteria Tobramycin- no Broth 4.00 Varied on plateUSP Lot L0E077 for each strain of bacteria ** Because of the highconcentration of this test antimicrobial, a 100x stock could not bemade. The calculations provided a 4x stock that is diluted in broth fromthe start and through the nine ½ dilutions.

E. Determination of MICs and MBCs

In the 96 well microtiter plate, the inoculated wells were observedafter 20 hours of incubation at 35 degrees C. In each row, there is arange from high to low concentrations of the drug respective of the rowfrom left to right, each well ½ the concentration of the adjacent wellto the left. The first clear well in the series was called the MIC. Thisis the concentration of drug that inhibited the growth of the bacteriafrom turning the broth turbid. Because of the nanoemulsions intrinsicturbidity, alamar blue (CellTiter Blue, Promega) was added for the rowcontaining nanoemulsion at a rate of 20 uL to 100 uL of culture volume.The plate was returned to the incubator for one hour and the first bluewell was called the MIC (wells with bacterial growth turned pink atlower concentrations of nanoemulsion). The starting inoculum in eachwell was generally between 2-7×10̂5 cfu/mL. The specific concentrationfor starting inoculum was determined for each test plate by sampling theinoculum used to inoculate the microtiter plate at the time it was setup. The exact concentration of bacteria was used to determine the MBCfor each drug in the panel for each test plate. The MBC is defined asthe concentration of drug that reduced the number of bacteria 99.9%. TheMIC is the landmark used to begin sampling 10 uL/well from the MIC plusfour wells to the left and put on agar plates, one sample per plate. Theagar plates were incubated for 24 hours at 35 degrees C. and colonieswere counted. The resulting colony counts were compared to the startingconcentration of the inoculum to determine which well, that is whichconcentration of drug, resulted in a three log drop in cfu/mL.

F. Checkerboard Synergy Study

The starting inoculum in each well was generally between 2-7×10̂5 cfu/mL.The specific concentration for starting inoculum was determined for eachtest plate by sampling the inoculum used to inoculate the microtiterplate at the time it was set up. Those exact concentrations of bacteriawere used to determine the MBC for each drug in the panel for each testplate. The MBC is defined as the concentration of drug that reduced thenumber of bacteria 99.9%, a three log drop from the originalconcentration of bacteria. The MIC is the landmark used to beginsampling 10 uL/well from the MIC plus all wells through to column 11 tothe right and put on agar plates, one sample per plate. The agar plateswere incubated for 24 hours at 35 degrees C. and colonies were counted.The resulting colony counts were compared to the starting concentrationof the inoculum to determine which well, that is which concentration ofdrug, resulted in a three log drop in cfu/mL.

In the 96 well microtiter plate, the inoculated wells were observedafter 20 hours of incubation at 35 degrees C. In each row, there is arange from high to low concentrations of the drug respective of the rowfrom right to left, each well ½ the concentration of the adjacent wellto the left. Typically, but not always, the rows contained thenanoemulsion, drug A. Going up and down in the columns is drug B andhere is the second drug tested in combination going high to low from Ato G, respectively. Row H did not contain the second drug for it to actas the control for drug A. Column 1 did not contain any drug A so thatcolumn 1 could be the control for drug B. Traditionally, first clearwell in the series from right to left in the rows was called the MIC.This is the concentration of drug that inhibited the growth of thebacteria from turning the broth turbid, in this case as indicated by acolor change from blue to pink since alamar blue was used to find themetabolic activity to define the MIC. To each well 20 uL of CellTiterBlue from Promega was added. That is 20% of the original culture volume.

The well with the MIC and the well with the MBC were noted for each row.All wells noted for the MIC of the two drugs in combination werecompared to the MIC of concentration of drug A and drug B each actingalone. For example, a well that contained 2 ug/mL of drug A and 8 ug/mLof drug B would have the concentration of A at 1 ug/mL when incombination divided by 16 ug/mL when acting alone. This renders a ratioof 0.125. Further in this example, 8 ug/mL of drug B would be divided by32 ug/mL of B acting alone. The ratio for drug B in combination:alone isthen 0.25. In this example the FIC index is said to be 0.125 for drug Aand 0.25 for drug B, the sum is 0.325 in this example. The definition ofsynergy is for the sum to be equal to 0.5 or less. Indifference isgreater than 0.5 to less than or equal to four. Antagonism is greaterthan four. See e.g., FIG. 19 for a detailed illustration.

G. Time-Kill Study

A bacterial suspension of Burkholderia NB 29 @ O.D. 0.14-0.20 was madeup. In a saline tube, 500 uL saline and 500 uL of the bacterialsuspension was mixed for the TO, non-treated control, then mixed one toone with TA (Trypton-Azolectin) broth with 5 mM CaCl₂ to be diluted andplated as described below. In a treatment tube, added one to one thebacterial suspension to the treatment mix (2×P4075EC at 64 ug/mL CPC and10 mM EDTA) and after 10, 20 and 30 minutes drew 400 uL at each timepoint and mixed with 400 uL TA broth with 5 mM CaCl₂. Next, 100 uL weredrawn to be diluted serially 1/10 in saline to 10⁻⁸. 100 uL were thendrawn from each dilution tube in the series and plated in triplicate.These plates were incubated at 35° C. overnight and counted to determinethe cfu/mL at each time point. Simultaneously at the time the sample ofuntreated or treated bacteria was added to the neutralization TAsolution, a sample of 450 uL was added to 113 uL of glutaraldehyde foreach respective time point.

H. Scanning Electron Microscopy (SEM)

For scanning electron microscopy (SEM), 113 ul of 10% aqueous solutionof gluteraldehyde in Sorenson's buffer, pH 7.4, was mixed with 450 uL ofthe bacterial suspension that underwent the exposure to nanoemulsion.Mixtures were vortexed and placed at 4° C. for at least 18 hours. Table19 provides the procedure for fixing and staining the samples forscanning electron microscopy.

TABLE 19 Method for fixing and staining samples for SEM Step 1 Sampleswere fixed in 2.5% glutaraldehyde in Sorenson's buffer, pH 7.4 thenagitated on a rotary stirrer at each step 2 Samples were rinsed twicefor 15 minutes each in 0.1 Sorensen's buffer 3 samples were fixed in1.0% OsO₄ in Sorenson's buffer 4 samples were rinsed twice for 5 minuteseach in 0.1M Sorenson's buffer 5 Samples were dehydrated for 15 minuteseach in each of the following: 30% EtOH, 50% EtOH, 70% EtOH, 90% EtOH,100% EtOH, 100% EtOH 6 Samples were immersed in four, 15 minutes changesof hexamethyldisilazane (HMDS) 7 Samples were removed following thefourth change of HMDS and replaced with just enough HMDS to covertissue. Samples were allowed to evaporate in the hood overnight 8Samples were mounted on SEM stubs, using the mixture of Colloidalgraphite and duco cement 9 Samples were placed in a vacuum desiccatorovernight 10 Samples were sputter-coated with gold using “Polaron”sputter coater 11 Samples were examined on an “Amray 1910 FE” SacnningElectron Microscope and digitally imaged using “Xstream” imagingsoftware

I. Results

a. MIC and MBC Data

The MIC₉₀/MBC₉₀ values for P₄₀₇5EC were 8/64 μg/ml for P. aeruginosa,64/>514 μg/ml for B. cenocepacia, 8/64 μg/ml for A. baumannii and 8/32μg/ml for S. maltophilia. Colistin had MIC₉₀/MBC₉₀ values of2/8, >32/>32, 1/>16 and >32/>32 for P. aeruginosa, B. cenocepacia, A.baumannii and S. maltophilia, respectively. Cefepime, imipenem,levofloxacin and tobramycin had MIC₉₀/MBC₉₀ values of ≧32/>32, ≧32/>32,16/16 and >32/>32 μg/ml, respectively, against all strains.

b. Synergy Data

Ten strains of Burkholderia, Stenotrophomonas and 10 strains ofAcinetobacter were tested to determine a shift in MIC when P₄₀₇5EC+EDTAwas in combination with either colistin or tobramycin, two traditionalantimicrobials used in the lungs of CF patients to treat chronic lunginfections. P₄₀₇5EC+EDTA in combination with colistin was found to besynergistic for 90% (in terms of the FIC) and 70% (in terms of the FBC)of the Stenotrophomonas strains, but indifferent, only 20% synergy in bythe FIC and 0% by the FBC, when in combination with tobramycin. For theAcinetobacter strains, P₄₀₇5EC+EDTA in combination with colistin wasfound to be indifferent, only 20% synergy in by the FIC and 0% by theFBC, as well as when in combination with tobramycin, only 10% synergy inby the FIC and 10% by the FBC. For the Burkholderia strains,P₄₀₇5EC+EDTA in combination with colistin was found to be indifferent,only 30% synergy in by the FIC and 10% by the FBC, but when incombination with tobramycin, 50% synergy in by the FIC and 20% by theFBC.

c. Time-Kill Study

Time-kill resulted in an overall 4.44 log reduction in cfu/ml from theuntreated beginning to the 30 minute time point. Each 10 minute timepoint had between 1-2 log reduction as follows: from the untreated tothe 10 minute point there was a 1.40 log reduction, from the 10 to 20minute time points there was a 1.91 log reduction and from the 20 to 30minute time points there was a 1.13 log reduction. See FIGS. 20A-20D.

TABLE 20 A comparison of MIC (μg/ml) of P₄₀₇5EC and comparator drugs.MIC & MBC 50 & 90 CF Pseudomonas Isolates All Drug Values in ug/mLP407-5EC & 5 mM P407-5EC & 0 mM Pseudo- Cefepime Tobramycin CefoxitinColistin Levofloxacin Imipenem Ceftazidime Pipercillin EDTA EDTA countmonas MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBCMIC MBC MIC MBC 1 NB1 2 8 1 16 >256 >256 2 >8 0.5 1 1 8 n/a n/a 10241024 16.0625 64.25 n/a 2 NB2 0.13 0.125 0.25 2 >256 >256 1 8 0.5 0.5 2 2n/a n/a 128 128 8.03125 32.125 n/a 3 NB3 1 2 0.5 4 >256 >256 1 8 0.250.5 2 16 n/a n/a >1024 >1024 4.015625 16.0625 n/a 4 NB4 1 1 0.54 >256 >256 2 4 0.25 0.5 4 8 n/a n/a 512 1024 4.015625 16.0625 n/a 5 NB52 4 0.5 4 >256 >256 2 8 0.5 1 2 16 n/a n/a >1024 >1024 8.03125 32.125n/a 6 NB6 2 4 0.5 4 >256 >256 2 4 0.5 1 1 2 n/a n/a 1024 >1024 4.01562516.0625 n/a 7 NB7 2 2 1 >16 >256 >256 2 4 0.5 1 4 16 n/a n/a 512 10244.015625 16.0625 n/a 8 NB8 2 2 1 8 >256 >256 2 8 0.5 0.5 4 4 n/an/a >1024 >1024 4.015625 8.03125 n/a 9 NB9 2 2 1 8 >256 >256 2 8 0.5 0.54 >32 n/a n/a 512 >1024 4.015625 64.25 n/a 10 NB10 2 2 0.5 4 >256 >256 22 0.5 0.5 4 8 n/a n/a 256 512 8.03125 64.25 n/a 11 NB11 16 32 24 >256 >256 1 4 8 16 2 8 n/a n/a n/a n/a <4.015625 16.0625 n/a 12 NB12 432 0.5 8 >256 >256 4 8 0.25 1 >32 >32 256 512 n/a n/a <4.015625 16.0625514 >2056 13 NB13 4 16 1 4 >256 >256 2 8 0.5 2 8 16 512 512 n/a n/a8.03125 16.0625 514 >2056 14 NB14 32 >32 32 >32 >256 >256 2 416 >16 >32 >32 1024 >1024 n/a n/a <4.015625 32.156 4.01563 >64.25 15NB15 8 >32 0.5 8 >256 >256 2 8 2 4 8 >32 256 512 n/a n/a <4.01562516.0625 1028 >2056 16 NB16 16 16 2 16 >256 >256 2 4 2 8 8 32 256 >1024n/a n/a <4.015625 >64.25 2056 >2056 17 NB17 >32 >32 16 32 >256 >256 1 416 16 >32 >32 n/a n/a n/a n/a <4.015625 16.0625 n/a 18 NB18 >32 >3216 >32 >256 >256 1 4 4 8 >32 >32 n/a n/a n/a n/a <4.015625 <4.015625 n/a19 NB19 16 32 2 16 >256 >256 2 4 2 4 >32 >32 512 1024 n/a n/a <4.0156258.03125 1028 >2056 20 NB20 8 8 0.5 8 >256 >256 2 >8 0.5 1 2 16 256 512n/a n/a 8.03125 8.03125 1028 >2056 MIC Cefe- 2 Tobramycin 1Cefoxitin >256 Colistin 2 Levo- 0.5 Imipenem 4 Ceftaz- n/a Pipercillinn/a P407-5EC 4.015625 P407- n/a 50 pime floxacin idime & 5 mM 5EC & EDTA0 mM EDTA MIC 90 32 16 >256 2 8 >32 n/a n/a 8.03125 n/a MBC Cefe- 8Tobramycin 8 Cefoxitin >256 Colistin 4 Levo- 1 Imipenem 16 Ceftaz- n/aPipercillin n/a P407-5EC 16.0625 P407- n/a 50 pime floxacin idime & 5 mM5EC & EDTA 0 mM EDTA MBC >32 >16 >256 8 16 >32 n/a n/a 64.25 n/a 90 MIC& MBC 50 & 90 CF Burkholderia Isolates All Drug Values in ug/mL P407-5EC& 5 mM Cefepime Colistin Imipenem Levofloxacin Ceftazidime TobramycinCefoxitin EDTA count Burkholderia MIC MBC MIC MBC MIC MBC MIC MBC MICMBC MIC MBC MIC MBC MIC MBC 1 NB21 32 >32 >32 >32 16 16 2 2 8 >32 32 >32256 >256 32.125 514 2 NB22 >32 >32 >32 >32 16 16 1 4 8 >32 >32 >32 128256 32.125 >514 3 NB23 >32 >32 >32 >32 >32 >328 >16 >32 >32 >32 >32 >256 >256 <4.015625 64.25 4 NB24 32 >32 >32 >3232 >32 8 16 4 32 >32 >32 128 >256 32.125 514 5 NB25 >32 >32 >32 >3232 >32 4 8 16 32 >32 >32 128 256 64.25 257 6 NB26 >32 >32 32 >32 >32 >328 8 16 32 >32 >32 128 256 <4.01563 64.25 7 NB27 >32 >32 >32 >32 16 >32 48 8 16 >32 >32 128 >256 32.125 257 8 NB29 8 >32 >32 >32 8 16 1 44 >32 >32 >32 256 >256 16.0625 >514 9 NB30 8 16 >32 >32 4 4 1 1 2 2 3232 128 128 <4.015625 >64.25 10 NB28b 4 4 >32 >32 0.5 0.5 1 2 16 16 1 416 16 <4.015625 >64.25 MIC 50 Cefepime 32 Colistin >32 Imipenem 16Levofloxacin 2 Ceftazidime 8 Tobramycin >32 Cefoxitin 128 P407-5EC & 5mM 16.0625 EDTA MIC 90 >32 >32 >32 8 16 >32 256 64.25 MBC 50Cefepime >32 Colistin >32 Imipenem 32 Levofloxacin 4 Ceftazidime 32Tobramycin >32 Cefoxitin 256 P407-5EC & 5 mM 128.5 EDTA MBC90 >32 >32 >32 >16 >32 >32 >256 >514 MIC & MBC 50 & 90 CF AcinetobacterIsolates All Drug Values in ug/mL Cefepime Colistin ImipenemLevofloxacin Ceftazidime Tobramycin Cefoxitin P407-5EC & 1 mM EDTA countAcinetobacter MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBCMIC MBC 1 NB31 16 16 1 1 1 1 16 16 >32 >32 1 1 >256 >256 4 16 2 NB3232 >32 1 8 32 32 8 8 >32 >32 1 4 256 256 4 64 3 NB33 >32 >32 1 4 32 >328 8 >32 >32 2 8 >256 >256 4 64 4 NB34 32 32 1 1 32 32 16 16 32 3216 >32 >256 >256 4 8 5 NB35 32 >32 1 4 8 8 >16 >16 >32 >32 216 >256 >256 8 32 6 NB36 2 2 1 1 0.125 0.125 1 1 2 4 0.5 0.5 128 128 216 7 NB37 32 >32 1 >16 4 8 16 16 32 32 0.5 2 >256 >256 2 16 8NB38 >32 >32 1 >16 32 >32 8 >16 >32 >32 1 16 >256 >256 4 64 9 NB39 >3232 1 8 2 2 16 16 32 >32 2 4 >256 >256 2 16 10 NB40 >32 >32 1 >16 232 >16 >16 >32 >32 >32 >32 >256 >256 8 >128 MIC 50 Cefepime 32 Colistin1 Imipenem 4 Levofloxacin 16 Ceftazidime >32 Tobramycin 1 Cefoxitin >256P407-5EC & 1 mM 4 EDTA MIC 90 >32 1 32 >16 >32 16 >256 8 MBC 50Cefepime >32 Colistin 4 Imipenem 8 Levofloxacin 16 Ceftazidime >32Tobramycin 4 Cefoxitin >256 P407-5EC & 1 mM 16 EDTA MBC90 >32 >16 >32 >16 >32 >32 >256 64 MIC & MBC 50 & 90 CF StenotrophomonasIsolates All Drug Values in ug/mL Cefepime Colistin ImipenemLevofloxacin Ceftazidime Tobramycin Cefoxitin P407-5EC & 1 mM EDTA countStenotrophomonas MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBCMIC MBC 1 NB41 32 >32 2 32 >32 >32 2 4 >32 >32 >32 >32 >256 >256 8 8 2NB42 >32 >32 2 8 >32 >32 8 16 8 >32 32 >32 >256 >256 16 16 3NB43 >32 >32 16 >32 >32 >32 2 4 >32 >32 >32 >32 >256 >256 8 16 4NB44 >32 >32 32 >32 >32 >32 8 16 >32 >32 >32 >32 256 >256 <1 16 5 NB4532 >32 8 16 >32 >32 1 2 >32 >32 >32 >32 >256 >256 8 16 6 Steno1 >32 >32 >32 >32 >32 >32 1 4 >32 >32 >32 >32 >256 >256 8 16 7 Steno2 >32 >32 32 >32 32 >32 4 8 >32 >32 >32 >32 >256 >256 4 16 8 Steno3 >32 >32 >32 >32 >32 >32 1 4 >32 >32 >32 >32 256 >256 8 32 9 Steno 416 >32 1 4 >32 >32 0.5 2 2 16 >32 >32 256 256 2 32 10 Steno 6 >32 >3216 >32 32 32 2 4 32 >32 16 32 128 256 8 32 11 Steno 8 >32 >32 832 >32 >32 2 2 16 >32 >32 >32 >256 >256 8 16 MIC 50 Cefepime >32Colistin 16 Imipenem >32 Levofloxacin 2 Ceftazidime >32 Tobramycin >32Cefoxitin >256 P407-5EC & 1 mM 8 EDTA MIC 90 >32 >32 >32 8 >32 >32 >2568 MBC 50 Cefepime >32 Colistin >32 Imipenem >32 Levofloxacin 4Ceftazidime >32 Tobramycin >32 Cefoxitin >256 P407-5EC & 1 mM 16 EDTAMBC 90 >32 >32 >32 16 >32 >32 >256 32

TABLE 21 Collapsed Summary of Data Table 20 P407-5EC & Cefepime ColistinImipenem Levofloxacin Ceftazidime Tobramycin Cefoxitin 5 mM EDTAPseudomonas MIC 50 2 2 4 0.5 Not Tested 1 >256 4.015625 MIC 90 32 2 >328 Not Tested 16 >256 8.03125 MBC 50 8 4 16 1 Not Tested 8 >256 16.0625MBC 90 >32 8 >32 16 Not Tested >16 >256 64.25 Burkholderia MIC 5032 >32 >32 2 8 >32 128 16.0625 MIC 90 >32 >32 >32 8 16 >32 256 64.25 MBC50 >32 >32 32 4 32 >32 256 128.5 MBC90 >32 >32 >32 >16 >32 >32 >256 >514 Acinetobacter MIC 50 32 1 4 16 >321 >256 4 MIC 90 >32 1 32 >16 >32 16 >256 8 MBC 50 >32 4 8 16 >32 4 >25616 MBC 90 >32 >16 >32 >16 >32 >32 >256 64 Stenotrophomonas MIC 50 >3216 >32 2 >32 >32 >256 8 MIC 90 >32 >32 >32 8 >32 >32 >256 8 MBC50 >32 >32 >32 4 >32 >32 >256 16 MBC 90 >32 >32 >32 16 >32 >32 >256 32Isolate FIC Synergy FBC Synergy Isolate FIC Synergy FBC SynergyCheckerboard Synergy Study Results; Acinetobacter NB31 Colistin 0.83indifferent 2.44 indifferent NB31 Tobra 0.98 indifferent 1.50 no NB32Colistin 0.58 indifferent 1.36 indifferent NB32 Tobra 0.88 indifferent1.79 no NB33 Colistin 0.33 yes 2 indifferent NB33 Tobra 1.27 indifferent1.75 no NB34 Colistin 0.46 yes 1.10 indifferent NB34 Tobra 0.41 yes 0.24yes NB35 Colistin 0.72 indifferent 0.97 indifferent NB35 Tobra 1.21indifferent 1.44 no NB36 Colistin 0.56 indifferent 2 indifferent NB36Tobra 3.30 indifferent 2.29 no NB37 Colistin 0.63 indifferent 1.00indifferent NB37 Tobra 0.75 indifferent 1.72 no NB38 Colistin 0.77indifferent 0.68 indifferent NB38 Tobra 1.05 indifferent 1.57 no NB39Colistin 0.72 indifferent 1.22 indifferent NB39 Tobra 3.30 indifferent3.52 no NB40 Colistin 0.63 indifferent 0.91 indifferent NB40 Tobra 3.01indifferent 3.01 no 20% 0% 10% 10% Checkerboard Synergy Study Results;Stenotrophomonas NB42 Colistin 0.08 yes 1.28 indifferent NB42 Tobra 0.14yes 2.15 indifferent NB43 Colistin 0.08 yes 0.15 yes NB43 Tobra 1.40indifferent 2.29 indifferent NB44 Colistin 1.01 indifferent 0.57indifferent NB44 Tobra 1.5 indifferent 2.86 indifferent NB45 Colistin0.04 yes 0.45 yes NB45 Tobra 0.97 indifferent 1.50 indifferent Steno 1Colistin 0.08 yes 0.11 yes Steno 1 Tobra 0.79 indifferent 1.57indifferent Steno 2 Colistin 0.09 yes 0.04 yes Steno 2 Tobra 0.04 yes2.23 indifferent Steno 3 Colistin 0.09 yes 0.14 yes Steno 3 Tobra 0.79indifferent 1.29 indifferent Steno 4 Colistin 0.46 yes 1.14 indifferentSteno 4 Tobra 1.72 indifferent 1.61 indifferent Steno 6 Colistin 0.09yes 0.02 yes Steno 6 Tobra 0.59 indifferent 0.89 indifferent Steno 8Colistin 0.09 yes 0.07 yes Steno 8 Tobra 0.63 indifferent 1.15indifferent 90% 70% 20% 0% Checkerboard Synergy Study Results;Burkholderia NB21 Colistin 1.45 indifferent 2.07 indifferent NB21 Tobra0.27 yes 0.48 yes NB22 Colistin 1.37 indifferent 2.29 indifferentNB22Tobra 0.35 yes 1.23 indifferent NB23 Colistin 1.01 indifferent 2.00indifferent NB23 Tobra 1.06 indifferent 1.09 indifferent NB24 Colistin1.72 indifferent 2.00 indifferent NB24 Tobra 1.31 indifferent 1.36indifferent NB25 Colistin 2.00 indifferent 3.00 indifferent NB25 Tobra1.45 indifferent 0.99 indifferent NB26 Colistin 0.56 yes 0.04 yes NB26Tobra 0.12 yes 0.92 indifferent NB27 Colistin 1.93 indifferent 1.64indifferent NB27 Tobra 1.44 indifferent 1.27 indifferent NB28b Colistin0.29 yes 1.54 indifferent NB28b Tobra 0.54 yes 1.06 indifferent NB29Colistin 1.61 indifferent 1.68 indifferent NB29 Tobra 0.32 yes 0.07 yesNB30 Colistin 0.02 yes 1.86 indifferent NB30 Tobra 0.27 yes 0.54 yes 30%10% 50% 30%

TABLE 22 Summary of Synergy Data with Comparison to Original MIC/MBCData of Each Drug Alone P₄₀₇5EC & Stenotrophomonas Colistin Tobramycin 1mM EDTA MIC 50 16 >32 8 MBC 50 >32 >32 16 MIC 90 >32 >32 8 MBC90 >32 >32 32 % FIC Synergy 90% 20% % FBC Synergy 70%  0% Synergy NoInterference P₄₀₇5EC & Acinetobacter Colistin Tobramycin 1 mM EDTA MIC50 1 1 4 MBC 50 4 4 16 MIC 90 1 16 8 MBC 90 >16 >32 64 % FIC Synergy 20%10% % FBC Synergy  0% 10% No Interference No Interference P₄₀₇5EC &Burkholderia Colistin Tobramycin 5 mM EDTA MIC 50 >32 >32 16 MBC50 >32 >32 64 MIC 90 >32 >32 128 MBC 90 >32 >32 >514 % FIC Synergy 30%50% % FBC Synergy 10% 30% No Interference No Interference

TABLE 23 Time Kill with additional 5 mM EDTA Pseudomonas with W205EC at4 ug/mL + 5 mM EDTA, 1 min resulted in a 2 log drop in count.Burkholderia Burkholderia MIC 50 = 16 ug/mL and 90 = 64 ug/mL NB29 MIC =16 32 ug/mL P4075EC + 5mM EDTA Starting Bacteria at 0.159 O.D. at 625 nm= ----8.24 × 10{circumflex over ( )}7 cfu/mL Set 0 min Set Set SetAverage (Saline) 8.24E+07 Average 10 min 3.27E+06 Average 20 min4.00E+04 Average 30 min 2.99E+03 0 min (Saline) 10 min 20 min 30 min SetA cfu/mL Set A cfu/mL Set A cfu/mL Set A cfu/mL 2.00E+09 0 2.00E+09 02.00E+09 0 2.00E+09 0 2.00E+08 0 2.00E+08 0 2.00E+08 0 2.00E+08 02.00E+07 5 1.00E+08 2.00E+07 0 2.00E+07 0 2.00E+07 0 2.00E+06 448.80E+07 2.00E+06 0 2.00E+06 0 2.00E+06 0 2.00E+05 128 2.56E+07 2.00E+0523 4.60E+06 2.00E+05 1 2.00E+05 0 2.00E+04 TNTC 2.00E+04 180 3.60E+062.00E+04 1 2.00E+04 2.00E+04 0 2.00E+03 Lawn 2.00E+03 TNTC 2.00E+03 193.80E+04 2.00E+03 2 4.00E+03 2.00E+02 Lawn 2.00E+02 TNTC 2.00E+02 1513.02E+04 2.00E+02 7 1.40E+03 2.00E+01 Lawn 2.00E+01 Lawn 2.00E+01 TNTC2.00E+01 134 2.68E+03 Average 7.12E+07 Average 4.10E+06 Average 2.94E+04Average 2.69E+03 Set B cfu/mL Set B cfu/mL Set B cfu/mL Set B cfu/mL2.00E+09 0 2.00E+09 0 2.00E+09 0 2.00E+09 0 2.00E+08 0 2.00E+08 02.00E+08 0 2.00E+08 0 2.00E+07 5 1.00E+08 2.00E+07 0 2.00E+07 0 2.00E+070 2.00E+06 39 7.80E+07 2.00E+06 1 2.00E+06 2.00E+06 0 2.00E+06 02.00E+05 234 4.68E+07 2.00E+05 11 2.20E+06 2.00E+05 1 2.00E+05 02.00E+04 TNTC 2.00E+04 155 3.10E+06 2.00E+04 6 1.20E+05 2.00E+04 02.00E+03 TNTC 2.00E+03 TNTC 2.00E+03 14 2.80E+04 2.00E+03 0 2.00E+02Lawn 2.00E+02 Lawn 2.00E+02 177 3.54E+04 2.00E+02 18 3.60E+03 2.00E+01Lawn 2.00E+01 Lawn 2.00E+01 TNTC 2.00E+01 128 2.56E+03 Average 7.49E+07Average 2.43E+06 Average 6.11E+04 Average 3.08E+03 Set C cfu/mL Set Ccfu/mL Set C cfu/mL Set C cfu/mL 2.00E+09 0 2.00E+09 0 2.00E+09 02.00E+09 0 2.00E+08 0 2.00E+08 0 2.00E+08 0 2.00E+08 0 2.00E+07 61.20E+08 2.00E+07 1 2.00E+07 0 2.00E+07 0 2.00E+06 41 8.20E+07 2.00E+061 2.00E+06 2.00E+06 0 2.00E+06 0 2.00E+05 TNTC 2.00E+05 22 4.40E+062.00E+05 0 2.00E+05 0 2.00E+04 TNTC 2.00E+04 172 3.44E+06 2.00E+04 12.00E+04 2.00E+04 0 2.00E+03 Lawn 2.00E+03 TNTC 2.00E+03 18 3.60E+042.00E+03 2 4.00E+03 2.00E+02 Lawn 2.00E+02 TNTC 2.00E+02 164 3.28E+042.00E+02 12 2.40E+03 2.00E+01 Lawn 2.00E+01 Lawn 2.00E+01 TNTC 2.00E+01161 3.22E+03 Average 1.01E+08 Average 3.28E+06 Average 2.96E+04 Average3.20E+03

J. Conclusions

This examples demonstrates that nanoemulsions, such as the testedP₄₀₇5EC, are effective against strains that are multidrug-resistant,including colistin-resistant isolates of Burkholderia andStenotrophomonas. None of the described lipid A modifications inPseudomonas species impacted the MIC/MBCs with P₄₀₇5EC. No evidence ofantagonism with two major antibiotics, colistin and tobramycin, wasobserved and in the case of the Stenotrophomonas, synergy was evident.This is valuable because the treatment of patients with CF should notneed their normal antibiotic regime suspended in order to use thenanoemulsion, complicating their treatment programs. The SEM imagesdemonstrate the kill on contact mechanism, here in this case a Gramnegative bacterium with a reputation of having a tough outer membrane.

Example 9 Topical Nanoemulsion Therapy Reduces Bacterial Wound Infectionand Inflammation Following Burn Injury Materials and Methods

Reagents.

Unless otherwise indicated, all reagents were purchased fromSigma-Aldrich Corp. (St. Louis, Mo.).

Animals.

Male specific pathogen-free Sprague-Dawley rats (Harlan, Indianapolis,Ind.) weighing approximately 250-300 g were used in all experiments.Rats were housed in standard cages and allowed to acclimate to theirsurroundings for 7 days prior to being used in experiments. They werekept on a 12 hour light cycle and provided with unrestricted access tostandard rat chow and water throughout the study. Experiments wereperformed in accordance with National Institutes of Health guidelinesfor care and use of animals. Approval for the experimental protocol wasobtained from the University of Michigan Animal Care and Use Committee.

Burn Model.

Animals were anesthetized with a 40 mg/kg intraperitoneal (ip) injectionof sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago,Ill.). Dorsal hair was closely clipped and removed using Nair depilatorycream (Church & Dwight Inc., Princeton, N.J.) resulting in a thoroughand uniform removal of fur. Each rat was placed in an insulated,custom-made mold, which exposes the dorsal region over 20% of the totalbody surface area. The burn surface area as a fraction of total bodysurface area was determined using Meeh's formula: body surface area(cm²)=9.46×(animal weight (g))^(2/3) (See Gilpin, Burns 22(8):607-611,1996). Partial thickness scald burn injury was achieved by placing theexposed skin of the rat in a 60° C. water bath for 27 seconds. Sham burnanimals received the same treatment except they were immersed in roomtemperature water (21-24° C.). The burn wound was scrub-débrided withdry sterile gauze and rinsed with 0.9% sterile NaCl. Each animal wasresuscitated with 4 mL Ringer's lactate/% total body surface areaburn/kg body weight. One half of this fluid volume was givenintraperitoneally and half subcutaneously immediately following the burninjury. The burn injured skin was left uncovered to air dry. Afterdrying, an occlusive dressing of sterile TELFA (Kendall Co., TycoHealthcare Group LP, Mansfield, Mass.) and TEGADERM HP (3M Health Care,St Paul, Minn.) was applied to prevent wound contamination. Duringexperiments each rat was singly housed and received 0.01 mg/kgbuprenorphine subcutaneously at the time of burn and at 16 hours forpost burn pain control.

Local Wound Treatment.

Stock nanoemulsion W₂₀5GBA₂ED was obtained from NanoBio Corporation (AnnArbor, Mich.). This nanoemulsion was manufactured by emulsification ofsuper-refined soybean oil and water with surfactants and alcohol asdescribed herein. The resultant droplets had a mean particle diameter of350 nm. The experimental solution was made by diluting 1 mL of the 60%stock formulation with 4.88 mL sterile saline and adding 120 μL of 1 Methylenediaminetetraacetic acid (EDTA) giving a final concentration of10% W₂₀5GBA₂ED and 20 mM EDTA. A placebo nanoemulsion (W₂₀5GBA₂EDplacebo) compound was manufactured in the same manner as W₂₀5GBA₂ED, butbenzalkonium chloride was omitted from the formulation. 5% Sulfamylon(UDL Laboratories, Inc., Rockford, Ill.) solution was formulated bymixing 50 g of mafenide acetate powder in 1 L of 0.9% sterile saline.The control reagent used was 0.9% sterile saline. Experimental groupsconsisted of sham, burn, burn+W₂₀5GBA₂ED, burn+bacteria+saline,burn+bacteria+placebo, burn+bacteria+W₂₀5GBA₂ED, andburn+bacteria+Sulfamylon. Sixteen hours following burn injury animalswere anesthetized with inhaled isoflurane. The occlusive dressing andTELFA was removed. Nanoemulsion (W₂₀5GBA₂ED), placebo, Sulfamylon orsterile saline was applied in a uniform fashion to the burn woundsurface using a spray bottle. Animals in the sham or burn group receivedno topical treatment, but did undergo dressing change under anesthesia.The burn wound was then redressed with TELFA and a TEGADERM occlusivedressing. This treatment and dressing change was repeated at 24 hoursfollowing burn injury.

Bacterial Culture and Inoculation.

Pseudomonas aeruginosa isolated from a human burn patient was previouslyprovided by the Department of Pathology at the University of Michigan.This bacterial isolate is sensitive to the topical agent Silvadene andSulfamylon. A bacterial inoculum was prepared by thawing an aliquot (0.5mL, stored in 50% skim milk at −80° C.) in 40 mL of Trypticase soy broth(Becton Dickinson, Franklin Lakes, N.J.) and grown overnight at 37° C.with constant shaking at 275 rpm. A sample of the resultingstationary-phase culture was transferred to 35 mL of fresh Trypticasesoy broth and incubated for 2.5 hours to reach the log-phase. Thissubculture was transferred to a 50 mL conical polystyrene tube andcentrifuged for 10 minutes at 4° C. and 880 g. The bacterial pellet waswashed with 0.9% sterile saline, and resuspended in 10 mL of ice-coldsaline. The optical density of the suspension was measured at 620 nm andbacterial concentration (colony forming units (CFU)/mL) calculated usingthe formula OD₆₂₀×2.5×10⁸. The bacterial suspension was diluted with0.9% sterile saline to a final concentration of 1×10⁶ CFU per 100 μL.Eight hours following burn injury animals were anesthetized with inhaledisoflurane. The rats then underwent topical application of 1×10⁶ CFUs oflog-phase Pseudomonas aeruginosa in 100 μL of sterile saline pipettedonto a piece of TELFA in a uniform fashion followed by coverage with aTEGADERM occlusive dressing.

Tissue Harvest.

Thirty-two hours after thermal injury the animals were sacrificed andskin tissue samples harvested using sterile technique Skin samples wereused immediately or frozen in liquid nitrogen.

Quantitation of Bacterial Wound Infection.

A 100 mg piece of excised skin tissue was mechanically homogenized in 1mL of 0.9 NaCl. This homogenate was then further diluted with 9 mL ofsterile saline. Serial dilutions were performed and skin homogenatesplated in triplicate on blood agar plates (Becton Dickinson, FranklinLakes, N.J.). Culture plates were incubated for 24 hours at 37° C. andCFUs counted.

Dermal Cytokine Analysis (ELISA).

A 100 mg sample of dorsal skin was homogenized in 1 mL of ice-cold lysisbuffer consisting of 50 mL of PBS and protease inhibitor (Complete X,Roche, Indianapolis, Ind.) and 50 μL of Triton X (Roche). Homogenateswere centrifuged at 3000 g for 5 minutes and the supernatants collectedand stored frozen at −80° C. until use. Rat IL1-β, IL-6, TNF-α, CINC-1,CINC-3, IL-10 and TGF-β were measured by sandwich enzyme-linkedimmunosorbent assay (ELISA) using antibodies and reagents from R&DSystems, Inc. (Minneapolis, Minn.). The assay was carried out in 96-wellmicroplates (Immunoplate Maxisorb, Nunc, Neptune, N.J.) according to thekit instructions and samples were read using a microplate reader (BiotekInstruments, Winooski, Vt.) at 450 nm with a wavelength correction of540 nm. Cytokine concentrations were determined using the plate readersoftware and a 7-point standard curve. Results were adjusted forprevious dilution and expressed as pg/mL.

Detection of neutrophil sequestration (Myeloperoxidase assay). 100 mg ofskin tissue was mechanically homogenized in 1 mL ice cold potassiumphosphate buffer consisting of 115 mM monobasic potassium phosphate(Sigma Aldrich, Milwaukee, Wis.). Homogenates were centrifuged at 3000 gfor 10 min at 4° C., the supernatants were removed and the pellets werere-suspended in 1 mL C-TAB buffer consisting of dibasic potassiumphosphate, cetyltrimethylammonium bromide, and acetic acid (SigmaAldrich, Milwaukee, Wis.). The suspensions were sonicated (BransonSonifier 250, Danbury, Conn.) on ice for 40 seconds. Homogenates werecentrifuged at 3000 g for 10 min at 4° C. and the supernatant collected.Supernatants were incubated in 60° C. water bath for 2 hours (ShakerBath, 2568; Forma Scientific, Marietta, Ohio). Samples were stored at−80° C. until needed or assayed immediately.

20 μL standards (Calbiochem, Gibbstown, N.J.) or samples were added to a96-well immunosorbent micro-plates (NUNC, Rochester, N.Y.), followed bythe addition of 155 μL of 20 mM TMB/DMF consisting of3,3′,5,5′-tetramethylbenzidine/N,N-dimethylformamide in 115 mM potassiumphosphate buffer (Fischer Scientific, Pittsburgh, Pa.) to each well. Thesamples were mixed well, after which 20 μL of 3 mM H₂O₂ was rapidlyadded to each well. The reaction was stopped immediately by adding 50μL/well of 0.061 mg/mL Catalase (Roche, Indianapolis, Ind.). The plateswere read using a microplate reader at 620 nm. Myeloperoxidase (MPO)concentrations were calculated using a linear standard curve andadjusted for previous dilution. The final concentrations were expressedas μg/mL.

Determination of Dermal Capillary Leak and Tissue Edema (Evans Blue).

Animals were anesthetized 90 minutes before tissue harvest. 50 mg/kgbody weight of 10% Evans blue (Merck KgaA, Darmstadt, Germany) wasinjected ip into the burned animal at time 30.5 hours following thermalinjury. At the tissue harvest time point animals were exsanguinated byincision of the inferior vena cava. Systemic Evans blue was washed outby inserting a 20 G angiocatheter into the apex of the left ventriclepast the aortic valve and into the ascending aorta. A total of fourtimes the blood volume (7.46 mL/100 g body weight) of 0.9 NaCl with 100units/mL heparin was used to flush the vasculature and administered viaa perfusion pump at a constant flow rate. By the end of the perfusingperiod the effluent from the right atrium had turned clear. Dorsal skinsamples were harvested and a 100 mg sample was placed in 4 mL 99.5%formamide in polyethylene tubes. Tubes were placed on a shaker at roomtemperature for 48 hours for Evans blue extraction. Supernatants werecollected and the absorbance read on a microplate reader at 620 nm.Concentrations were calculated from an Evans blue in formamide standardcurve. Results are expressed as micrograms of Evans blue per mg of skintissue.

Histology.

Skin samples were fixed in 10% buffered formalin and embedded inparaffin. Eight μm thick sections were affixed to slides,deparaffinized, and stained with hematoxylin and eosin to assessmorphologic changes.

Detection of Hair Follicle Cell Apoptosis (TUNEL Assay).

Animals were anesthetized and underwent creation of a 20% partialthickness scald burn wound or sham injury. Treatment groups consisted ofsham, burn+saline, burn+placebo, and burn+W₂₀5GBA₂ED. Treatment anddressing changes were performed at 0 and 8 hours post-burn. No bacterialinfection was created in this experiment. Full-thickness skin sampleswere taken from three locations across the entire burn wound at 12, and24 hours post thermal injury for determination of hair follicle cellapoptosis. There were four animals per treatment group per time sample.

Apoptosis was detected in situ with fluorescein based labeling of DNAstrand breaks using terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) assay (ApopTag, CHEMICON International, Inc, Temecula,Calif.). The three fresh skin samples for each animal were placed indisposable vinyl cryomolds filled with optimal cutting temperaturecompound (Sakura Finetek, U.S.A., Inc., Torrance, Calif.), and frozen at−80° C. until ready for use. Frozen embedded skin specimens were cutinto 4-mm-thick serial sections in a cryostat and collected onSuperfrost Plus glass slides (Fisher Scientific, Pittsburgh, PN).Sections were fixed in 1% paraformaldehyde in PBS overnight at 4° C. forindirect immunofluorescence according to the manufacturer'sinstructions. After 2 washes with PBS, sections were post-fixed in a 2:1solution of ethanol:acetic acid for 5 minutes at −20° C. Following 2additional washes with PBS, sections were incubated in equilibriumbuffer for 5 minutes. Sections were then incubated with terminaldeoxynucleotidyl transferase enzyme in a humidified chamber for 1 h at37° C. The reaction was terminated by rinsing the sections in astop/wash buffer. Sections were incubated in a humidified chamber atroom temperature with antidigoxigenin fluorescein (fluoresceinisothiocyanate, FITC) for 30 min and rinsed 3 times in PBS. Afteraspiration, sections were washed once with water and coverslips appliedusing ProLong Gold Antifade (Molecular Probes, Inc, Eugene, Oreg.),which included 4¹ 6-diamidino-2-phenylindole dihydrochloride (DAPI)counterstaining

The TUNEL assay slides were blinded to groups, and under the microscopeappropriate hair follicle cells in a randomly chosen high-power fieldwere identified. Appropriate hair follicles for analysis were thosesectioned in the mid-sagittal or mid-coronal plane. A total of 3-6 hairfollicles were selected from among the three skin samples present on aslide. Fluorescent-labeled TUNEL slides were captured digitally atidentical time post-labeling to control fading of fluorescence using anOlympus BX-51 fluorescence microscope at fixed image capture settingsand 40× magnification. Each hair follicle was selected and firstdigitally captured by visualizing counterstained nuclei present usingthe DAPI excitation/emission channel. Then for each hair follicleanalyzed, the excitation/filter channel was changed to visualize thefluorescein-labeled TUNEL-positive cells, and images again digitallycaptured. Within the captured images a region of interest (ROI) wasdigitally defined, set to include only hair follicle cells and excludebright fluorescing hair shafts and surrounding cells (NIH Image Jsoftware, NIH, Bethesda, Md.). Fluorescence of TUNEL-positive cells wasquantified, normalized to ROI size, and expressed as pixels/areafraction, controlling for differences in ROI size.

Statistical Methods.

Statistical analysis and graphs were performed using GraphPad Prism 5.0software (GraphPad Software, La Jolla, Calif.). Results are presented asmean values±the SEM unless otherwise noted. Continuous variables wereanalyzed using an unpaired two-tailed Student's t-test and/or One-wayANOVA followed by Tukeys post-test comparisons. The Kruskal-Wallis testwith Dunn's multiple comparisons was used to evaluate differences inmedians for data with a non-parametric distribution. Discrete variableswere compared using Fisher's exact test. Statistical significance wasdefined as a p-value <0.05.

Topical Application of Nanoemulsion Reduces P. aeruginosa Growth in BurnWounds

Animals treated with nanoemulsion had a decreased mean (6.5×10⁴ vs.7.9×10⁷, p=0.07) and median (0 vs. 4.4×10⁶, p<0.05) number of CFUs ofbacteria per gram of skin tissue when compared to the saline treatedcontrols (See FIG. 21) A similar reduction in skin bacterial counts wasfound for W₂₀5GBA₂ED treated animals vs. those treated with theW₂₀5GBA₂ED placebo (mean: 6.5×10⁴ vs. 5.5×10⁶, p=0.02). When performingquantitative wound culture on clinical tissue samples a positive resultis generally considered to be growth of organisms at greater than 1×10⁵CFUs per g of tissue (See, e.g., Neal et al., J Burn Care Rehabil2:35-39, 1981; Taddonio et al., Burns Incl Therm Inj 14(3):180-184,1988; Uppal et al., Burns 33(4):460-463, 2007). Using these criteria, 29of 32 animals in the control group exhibited evidence of a positivequantitative wound culture and only 3 of 23 animals in the nanoemulsiongroup demonstrated proof of this level of wound infection (91% vs. 13%,p<0.0001, See Table 24, below). The Sulfamylon treated animals alsodemonstrated a significant reduction in the median wound bacterial levelwhen compared to the saline controls (3×10⁴ vs. 4.4×10⁶, p<0.05).Treatment with nanoemulsion or Sulfamylon produced a similar reductionin the level of Pseudomonas cultured from the burn wound when comparedto the saline treated animals. However, there was no statisticallysignificant difference between the placebo and Sulfamylon groups whereasthere was a difference for the W₂₀5GBA₂ED group compared to theW₂₀5GBA₂ED placebo.

TABLE 24 Quantitative Wound Culture Results Saline Placebo NB-201Sulfamyton Total Animals, n 32 12 23 10 Animals with CFU's/g > 1 × 29(91) 9 (75) 3 (13)* 2 (20)* 10⁵, n (%) *p < 0.05, vs. saline control,Fishers exact test

Nanoemulsion Treatment Following Burn Injury Attenuates DermalPro-Inflammatory Cytokine Levels

Scald injury resulting in a partial thickness burn produced differencesin dermal levels of IL-1β and cytokine-induced neutrophilchemoattractant-3 (CINC-3) within skin homogenates obtained 32 hourspost-injury compared to sham injured animals (See FIGS. 22A and E).Treatment with W₂₀5GBA₂ED at 16 and 24 hours post-burn reduced thedermal level of these two inflammatory mediators back down to thebaseline (sham) in the absence of bacterial infection. In experimentswhere a bacterial wound infection was not created, a difference inneutrophil sequestration as measured by myeloperoxidase assay was notobserved despite the rise in the rat CXC chemokine CINC-3 within burnedskin. A difference was found between all three groups (sham, burn, andburn+W₂₀5GBA₂ED) for CINC-1 (p=0.04, ANOVA), however the values forintergroup comparison did not reach statistical significance.

Nanoemulsion Treatment Following Burn Wound Infection with P. aeruginosaAttenuates Dermal Cytokine Levels and Results in Reduced NeutrophilSequestration

Skin homogenates from the nanoemulsion treated group had levels of IL-1βand IL-6 that were considerably diminished when compared to the levelsmeasured in the saline treated animals (See FIGS. 23A and B). There wasno statistically significant difference seen in the level of TNF-αbetween the two experimental groups of animals (See FIGS. 22 C and D).Treatment of the infected burn wound with Sulfamylon did not result inany significant alteration of dermal levels of the measuredproinflammatory cytokines (IL-1β, IL-6, TNF-α, CINC-1 or CINC-3) whencompared to controls. Treatment with either W₂₀5GBA₂ED or Sulfamylonreduced the level of myeloperoxidase found in the infected burn wound at32 hours post-injury. Thus, in some embodiments, treatment with ananoemulsion and/or antimicrobial reduces the level of neutrophilsequestration into a partial thickness burn wound.

Burn injury caused a rise in the level of the anti-inflammatory cytokineTGF-β, but not IL-10 when compared to the sham injured animals (See FIG.24). W₂₀5GBA₂ED treatment reduced the amount of TGF-β present in theinfected burn wound as compared to the level found in the burn woundalone. Accordingly, in some embodiments, the present invention providesthat W₂₀5GBA₂ED can be utilized to reduce acute burn wound dermalinflammation, and in further embodiments, can be utilized to reduce theeventual immunosuppression created by thermal injury.

On histological examination of skin from the saline control animals,there is loss of most of the epidermis and a diffuse cellular infiltratein the subepidermal region, extending into the lower dermal connectivetissue in which collagen fibrils are separated by the infiltratingleukocytes and edema fluid (See FIG. 25A). At a higher power, thecellular infiltrate between the collagen bundles consists almostentirely of neutrophils. Edema fluid causes separation of the collagenfibrils. In FIG. 25B, the skin was subjected to thermal injury followedby application of P. aeruginosa after which the nanoemulsion wastopically applied to the burned area. The keratin layers of theepidermis are separating and some of the keratin has been lost. There isa barely detectable intradermal presence of neutrophils together withneutrophils that are adhering to the wall of a venule, which has beenlongitudinally sectioned (in the center of the microphotograph). Thechanges in this microphotograph are substantially less extensive thanthose seen in FIG. 25A.

Quantification of Capillary Leak and Tissue Edema

Burn wounds are associated with significant levels of capillary leak.This can lead to depletion of the intravascular volume and a need forlarge amounts of intravenous crystalloid fluid administration. To assesswhether therapeutic treatment with a nanoemulsion reduced capillary leakin conjunction with reducing inflammation, an Evans blue assay wasutilized to measure vascular permeability. Quantitative measurement ofthe amount of this dye leaching out of the blood stream and into theskin tissue revealed that the nanoemulsion treated animals had lessevidence of post-burn capillary leak and tissue edema than the salinetreated controls (See FIG. 26).

Treatment with Nanoemulsion Reduces Burn Induced Hair-Follicle Apoptosis

Dermal apoptosis occurs in the hair follicle cells following thermalinjury. Using a fluorescence labeled TUNEL assay the burn wounds treatedwith saline showed evidence of intense FITC-TUNEL positive cells whichappear green (See FIG. 27). The DAPI nuclear stain allows identificationof coronal or sagittally sectioned hair follicles with the cellsstaining blue. FITC-TUNEL positive cells appear green and arerepresentative of apoptotic cells. In the merged images, the apoptotichair follicle cells are evident in slides from the burn+saline animalsand these changes are diminished in the burn+W₂₀5GBA₂ED treated animals.Counting the pixels of TUNEL positive cells within a hair follicleregion of interest allowed quantification of the reduction in hairfollicle cell apoptosis by treatment with topical nanoemulsion (See FIG.28). The saline treated control animals had an increased amount of TUNELpositive cells when compared to the sham burn animals. Both theW₂₀5GBA₂ED and placebo treatment resulted in a decrease in hair folliclecell apoptosis following partial thickness burn injury in tissueharvested 12 hours following thermal injury. This difference was notevident in the dermal skin sampled at 24 hours post-burn. Thus, in someembodiments, the present invention provides a method of reducingapoptotic cell death in hair follicles in the early post-burn periodcomprising administering a nanoemulsion (e.g., W₂₀5GBA₂ED) to a burnwound during an early post-burn period.

Example 10 Immunogenic Compositions Comprising Nanoemulsion andBurkholderia Immunogen and Methods of Inducing Protective ImmunityAgainst Burkholderia Species in a Subject Materials and Methods

B. cenocepacia Strain K56-2 Stock Maintenance and Culture. Burkholderiacenocepacia strain K56-2 was generously provided by Dr. Pam Sokol(University of Calgary). K56-2 is a clinical isolate and has been usedfor Burkholderia molecular microbiology and genomic studies (See, e.g.,Goldberg, 2007 Burkholderia Molecular Microbiology and Genomics. Gent,Belgium: Taylor & Francis). It is a representative of the transmissibleand virulent B. cenocepacia ET12 lineage (See, e.g., Johnson et al.,1994, J Clin Microbiol 32, 924-930; Saijan et al., 2008, Infect Immun76, 5447-5455). Burkholderia multivorans ATCC 17616 was used forcross-strain neutralization assays. Both K56-2 and ATCC 17616 strainswere stored at −80° C. in Luria-Bertani broth with 15% glycerol andrecovered from frozen stock on brain-heart infusion agar overnight at37° C.

B. cenocepacia Outer Membrane Protein (OMP) Preparation.

An overnight culture of K56-2 in brain-heart infusion media wascentrifuged at 3500 rpm for 20 minutes. The cell pellet was washedseveral times with PBS (pH 7.4), re-suspended in 1 mM EDTA in PBS (pH8.0), then incubated for 30 minutes at room temperature.Post-incubation, the bacteria were passed several times through a26-gauge needle (Becton Dickinson) using high pressure. Cell lysis wasachieved with Triton X-100 (Sigma) added to a final concentration of 2%and incubated for 10 minutes at room temperature. Mechanical separationwas performed with multiple rounds of sonication (1 minute each). Thesonicated lysates were then centrifuged twice for 10 minutes at 6000×g(Beckman Optima XL-100k ultracentrifuge, 25° C.) with the supernatantretained after each spin. Following the second centrifugation, thesupernatant was centrifuged at 100,000×g for 1 hour at 4° C. Theresulting pellet was resuspended in endotoxin-free PBS. The rough OMPpreparation was purified (endotoxin-depleted OMP) with anendotoxin-removal column (Pierce) according to the manufacturer'sinstructions. The flow-through fractions were stored at −80° C. untilused.

OMP Analysis.

The protein contained within the OMP preparation was quantified usingBCA Assay (Pierce). Western blotting and silver staining were performedaccording to established protocol as described previously (See, e.g.,Makidon et al., 2008, PLoS ONE 3, e2954). To quantify endotoxincontaminate, OMP was analyzed using the LAL Kinetic-QCL (Lonza).Endotoxin was detected at 1.93 endotoxin units/mg in theendotoxin-depleted OMP preparation. DNA contaminant removal was verifiedby agarose-EtBr gel electrophoresis and imaged on a UV table (See FIG.29A). Oligonucleotides contaminants were not detected in either thecrude OMP or the endotoxin-depleted OMP preparations.

OMP Sequencing and Verification.

Sample Preparation. The protein sample was separated by SDS-PAGE (SeeFIG. 29B). The protein band immunostained with the highest intensity(˜17 KDa) on western blot (See FIG. 29C) was excised and subjected toin-gel digestion. In-gel digestion was performed at the ProteinStructure Facility at the University of Michigan according to proceduresby Rosenfeld et al. and Hellman et al. See, e.g., Hellman et al., 1995,Analytical Biochemistry 224, 451-455; Rosenfeld et al., 1992, AnalyticalBiochemistry 203, 173-179). A gel slice containing 5-ρmol bovine serumalbumin (BSA) was analyzed in parallel as a positive control.

Mass Spectrometry.

HPLC-Electrospray Ionization (ESI) Tandem Mass Spectrometry (MS/MS) wasperformed on a Q-tof premier mass spectrometer (Waters Inc) fitted witha nanospray source (Waters Inc.). The mass spectrometer was calibratedwith a mass accuracy within 3 ppm. On-line capillary HPLC was performedusing a Waters UPLC system with an Atlantis C18 column (Waters, 100-mminner diameter, 100-mm length, 3-mm particle size). Digests weredesalted using an online trapping column (Waters, Symmetry C18, 180-mminner diameter, 20-mm length, 5-mm particle size) before being loadedonto the Atlantis column. A data-dependent tandem mass spectrometryapproach was utilized to identify peptides in which a full scan spectrum(survey scan) was used, followed by collision-induced dissociation (CID)mass spectra of all ions in the survey scan with a peak intensity risingabove 20 counts per second. The survey scan was acquired in V+ mode overa mass range of 50-2000 Da with lockmass correction (Gu-FibrinopeptideB, (M+H)²⁺: 785.8426) and charge state peak selection (2+, 3+ and 4+).The MS/MS scans were acquired for 5 seconds with collision energycontrol by charge state recognition.

Data Analysis and Bioinformatics.

Tandem mass spectra were acquired using Mass Lynx software (Version4.1). Raw data files were first processed for lockmass correction, noisereduction, centering, and deisotoping using the Protein Lynx GlobalServer software Ver. 4.25 (Waters Inc.). The generated peaklist filescontaining the fragment mass spectra were subjected to database searchesusing the ProteinLynxGlobal Server and Mascot (Matrix Science Inc.,Boston, Mass.) search engines and Swiss Prot and NCBI databases, as wellas the Burkholderia cenocepacia database downloaded from the SangerInstitute web site.

Preparation of Nanoemulsion-Based OMP (OMP-NE) Vaccine.

Nanoemulsion (W₈₀5EC: 5 vol. % of TWEEN 80, 8 vol. % of ethanol, 1 vol.% of CPC, 64 vol. % of soybean oil) and 22 vol. % of DiH₂O) was providedby NanoBio Corporation (Ann Arbor, Mich.), the droplets having aparticle diameter of 200-400 nm. OMP-NE formulations were prepared byvigorously mixing the endotoxin-depleted OMP preparation (See FIG. 29B,Lane F) with concentrated NE, using PBS as the diluent. For theintranasal immunizations, OMP-NEs were formulated to contain either 0.25μg/μL or 0.75 μg/uL OMPs mixed in 20% (v/v) NE.

Animals and Immunization Procedures.

Pathogen-free, outbred CD-1 mice (females 6-8 weeks old) were purchasedfrom Charles River Laboratories. The mice were housed in accordance tothe standards of the American Association for Accreditation ofLaboratory Animal Care. The use of these mice was approved by theUniversity of Michigan University Committee on Use and Care of Animals(UCUCA). The mice (n=10 per group) were vaccinated with twoadministrations of either OMP-NE vaccines four weeks apart. Intranasal(i.n.) immunizations were performed in mice anaesthetized withisoflurane using the IMPAC6 anesthesia delivery system. The anesthetizedanimals were held in a supine position and 20 μL (10 μL/nare) of OMP-NEvaccine was administered slowly to the nares using a micropipette tip.Mice were immunized with either 15 μg OMP with NE, 15 μg OMP in PBS, 5μg OMP with NE, or 5 μg OMP in PBS.

Phlebotomy, Bronchoalveolar Lavage, and Splenocyte Collection.

Blood was collected from the saphenous vein every 21 days throughout theduration of the study. The terminal sample was obtained by cardiacpuncture immediately following euthanasia. Whole blood samples wereseparated by centrifugation at 3500 rpm (15 minutes) followingcoagulation. The serum samples were stored at −20° C. until analyzed.Bronchoalveolar lavage (BAL) fluid was obtained from the miceimmediately following euthanasia as described (See Makidon et al., 2008,PLoS ONE 3, e2954). In vitro measurement of the cytokine response wasdetermined using the spleens of the vaccinated mice. Spleens weremechanically disrupted to obtain single-cell splenocyte suspension inPBS. Red blood cells were lysed with ACK buffer (150 mM NH4Cl, 10 mMKHCO3, 0.1 mM Na2EDTA) and removed by washing the cell suspension twicein PBS. The splenocytes were then resuspended in RPMI 1640 mediumsupplemented with 2% FBS, 200 nM L-glutamine, andpenicillin/streptomycin (100 U/mL and 100 mg/mL).

Enzyme-Linked Immunosorbent Assay (ELISA).

Serum IgG and mucosal secretory IgA (sIgA) were determined by ELISAagainst endotoxin-depleted OMP. The OMP was prepared at 15 μg/mL incoating buffer (Sigma) and 100 μL was applied per well to Polysorbplates (Nunc) for a 4° C. overnight incubation. Serum was seriallydiluted in 0.1% BSA/PBS. Either diluted serum or undiluted BAL was thenadded to the plates for an overnight incubation at 4° C. Following a 16hour incubation, the plates were washed and an alkaline phosphataseconjugated anti-mouse IgG (H&L), IgG1, IgG2a, IgG2b, sIgA (achain-specific) (Rockland Immunochemicals, Inc) antibody was added at1:2000 or 1:1000 in 0.1% BSA/PBS. The plates were read at OD₄₀₅ and endtiters based on naïve animal levels. Levels of sIgA were normalized tototal protein content.

Analysis of Antigens Recognized by Serum IgG.

In addition to western blot analysis, serum lipoprotein-specific andlipopolysaccharide-specific IgG antibodies in serum were measured usingELISA. Briefly, 96-well Polysorb assay plates (Nunc) were coated with0.05 μg/well of crude OMP preparation, endotoxin-depleted OMP, or LPS(List Biological Laboratories). Serum was collected from mice immunizedwith OMP+NE 8 weeks following prime vaccination. The serum was seriallydiluted in 0.1% BSA/PBS and then added to the coated wells. The ELISAprocedure was performed as described supra. To evaluate the immunogeniccontribution of protein and LPS contained within the OMP-based vaccine,an enzymatic protein digest was performed using 250 μg of PCR-graderecombinant proteinase K (Roche) in 100 μl of either the crude OMPpreparation or the endotoxin-depleted OMP preparation. Proteinase K wasincubated with the OMP preparations for 16 hours at room temperature.The protein digestion was verified using a silver-stained SDS-PAGE. LPSand OMP-protein specific epitopes were evaluated using western blotanalysis probing with serum from mice vaccinated with either OMP-NE orOMP in PBS, or probing with an anti-Pseudomonas mallei LPS antibody(GeneTex).

Analysis of Cytokine Expression.

Freshly isolated mouse murine splenocytes were seeded at 4×10⁶ cells/mL(RPMI 1640, 2% FBS) onto a 24-well plate and incubated for 72 hours withOMPs (15 μg/mL) or PHA-P mitogen (2 μg/mL) as a control. Cell culturesupernatants were collected and analyzed for the presence of cytokinesusing LUMINEX multi-analyte profiling beads (LUMINEX Corporation,Austin, Tex.), according to the manufacturer's instructions.

Measurement of Serum Antibody Neutralization.

B. cenocepacia strain K56-2 and B. multivorans strain ATCC 17616antibody-complement neutralization activity was assayed using serumharvested from mice 6 weeks following primary i.n. vaccination.Twenty-five μL of serum (serially diluted 1×, 2×, 3×, 4×, or 5×) wasadded to 25 μL of a solution containing 1×10³ K56-2 or B. multivoranssupplementing MH medium in microculture plates (Nunc). The plates wereincubated at 37° C. for 48 hours. Controls included 25 μL serum with 25μL of sterile 1×PBS and 50 μL of MH medium containing 1×10³ bacteria.Following incubation, the wells were mixed and 10 μL of solution wasplated on Burkholderia selective agar (BCSA) media (See Henry et al.,1997, J Clin Microbiol 35, 614-619) on polystyrene culture plates(Fisherbrand) then incubated 72 hours at 37° C. prior to manual colonyforming units (cfu) enumeration.

Pulmonary Bacterial Challenge.

Bacterial challenge studies were performed in immunized mice (n=5 pergroup) or non-vaccinated control mice (n=10) 10 weeks following primaryvaccination using a modified version of the chronic pulmonary model ofB. cepacia infection described (See Bertot et al., 2007, Infect Immun75, 2740-2752; Chu et al., 2002 Infect Immun 70, 2715-2720; Chu et al.,2004, Infect Immun 72, 6142-6147). Briefly, 5×10⁷ of K56-2 suspended in85 μL of PBS was instilled through a trans-tracheal catheter extendingto the bifurcation of the main-stem bronchi in anesthetized mice. Themice were maintained for a period of 6 days. Prior to the challengestudy, the mice were non-surgically implanted with programmabletemperature transponders (IPTT-3000, Bio Medic Data Systems, Inc.) fornon-invasive subcutaneous temperature measurement with a handheldportable scanner (DAS-6002, Bio Medic Data Systems, Inc.). In-lifeanalysis included monitoring the clinical status of each animal byevaluating the following metrics: body weight, body temperature, foodconsumption, and activity. Immediately following euthanasia, pulmonarytissues and the spleen were collected in sterile conditions and eachorgan was placed into individual containers containing 1 mL of 1×PBS(Mediatec Inc). The tissue was then homogenized using a Tissue TEAROR(Biospec Products Inc.) for 50 seconds on ice. Serial dilutions (1:10²to 1:10⁸) of the homogenate were plated onto separated BCSA plates. Allplates were then incubated for 72 hours at 37° C. prior to manual cfuenumeration. Whole blood was collected in tubes containing EDTA (BD)immediately following euthanasia. Anti-coagulated blood was processed todetermine total peripheral lymphocytes and mononuclear lymphocytes bythe Animal Diagnostic Laboratory at the University of Michigan, using aHEMAVETH 950 hematology analyzer (Drew Scientific, Inc.) in accordanceto manufacturer's recommendation.

Statistics.

Antibody end-titer results are expressed as mean±standard error of themean (SEM) or ±standard deviation (SD). The statistical significance wasdetermined by ANOVA (analysis of variance) using the Student t andFisher exact two-tailed tests. For the bacterial challenge studies, thedistribution of the colonization was highly skewed, and therefore thedata was transformed (natural log transformation) for datanormalization. The normalized mean bacterial response in the lung andspleen were compared using an independent samples test with a two sidedp-value. p<0.05 was considered to be statistically significant. Allanalyses were done with 95% confidence limits.

Identification of Immunoreactive Proteins

Immunoreactive OMP proteins were identified via silver stain (See FIG.29B) and western blot (See FIGS. 29C and D). Major reactive bands weredocumented at 62, 45, 17, and 10 KDa in both cases; however, theintensity of the bands was much higher with serum from mice immunizedwith the NE-based vaccine (See FIG. 29C) as compared to the blot probedwith an equivalent serum dilution from mice immunized with the OMP inPBS preparation (See FIG. 29D).

The band with the highest intensity was located at ˜17 KDa (See FIG.29C). For the purpose of identification, the 17 KDa band was isolatedfrom the gel and identified by MALDI-TOF analysis. The protein wasidentified as Burkholderia cepacia outer membrane lipoprotein A (OmpA)with a MW of 16.396 KDa (See, e.g., Ortega et al., 2005 J Bacteriol 187,1324-1333; Plesa et al., 2004, J Med Microbiol 53, 389-398). Sequenceanalysis using National Center for Biotechnology Information (NCBI)protein BLAST confirmed that these proteins are present in other closelyrelated Burkholderia species (See Table 25, below). A high degree ofprotein homology (87.9% sequence homology) was identified in numerousstrains of Burkholderia and Ralstonia organisms and highly conservedamino acids from the OmpA family residues are present in the 17 KDaprotein sequence (See Table 25). Thus, the present invention identifiedand provides immunoreactive OMP proteins from B. cepacia as well asproteins from related strains of Burkholderia species displaying a highdegree of homology to the identified immunoreactive OMP proteins from B.cepacia (e.g., that find use in immunogenic compositions comprising ananoemulsion (e.g., that are administered to a subject in order generatea specific immune response in the subject toward the immunogeniccomposition comprising nanoemulsion and immunoreactive OMP proteins orhomologues)).

TABLE 25 Protein sequence alignment of OmpA-like protein family.Identity Organisms Omp-A like sequences alignment (%) B.LDDKANAGAVSTOPSADNV SIYFDF DSYSVKDEYQPLLQQ QGNTDERSTSEYN ALALLGVVSLGKEKP LATGHDEASWA 100  centocepacia AQVNVDPLNDPNSPLAKR HAQYLKSHPQRHVLLALGQKRAEAVRR ADSQMEA QNRRADLVYQQ B. LDDKANAGAVSTQPSADNV SIYFDFDSYSVKDEYQPLLQQ QGNTDERSTSEYN ALALLGV VSLGKEKP LATGHDEASWA 100  dolosaAQVNVDPLNDPNSPLAKR HAQYLKSHPQRHVL LALGQKRAEAVRR ADSQMEA QNRRADLVYQQ B.LDDKANAGA+STQPSADNV SIYFDF DSYSVKDEYQPLLQQ QGNTDERSTSEYN ALALLGVVSLGKEKP LATGHDEASWA 98 multivorans AQVNVDPLNDPNSPLAKR HAQYLKSHPQRHVLLALGQKRAEAVRR ADSQMEA QNRRADLVYQQ B. LDDKANAGAVSTQPSADNV SIYFDFDSYSVKDEYQPL+QQ QGNTDERSTSEYN A+ALLGV VSLGKEKP LA+GHDEASWA 96 ambifariaAQVNVDPLNDPNSPLAKR HAQYLKSHPQRHVL LALGQKRAEAVRR DSQMEA QNRRALDVYQQ B.LDDKAN−AGAVSTQPSADN SIYFDF DSYSVKDEYQPLLQQ QGNTDERSTSEYN A+ALLGVVSLGKEKP LATGHDEASWA 97 vietnamiensis VAQVNRVDPLNDPNSPLAK HAQYLKSHPQRHVLLALGQKRAEAVRR DSQMEA QNRRADLVYQQ B. LD+KNAGA+STQP+ +NVA SIYFDFDSYSVKDEYQPL+QQ QGNTDERSTSEYN A+ALLGV VSLGKEKP LA GHDEASWA 92 ubanensisQV VDPLNDPNSPLAKR HAQYLKSHPQRHVL LALGQKRAEAVRR ADSQMEA QNRR+DLVYQQ B.LD+ AN−GAVSTQP+ +NA SIYFDF DSYSV+D+YQPLLQQ QGNTDERSTSEYN AL+LLGVVSLGKEKP LA GHDEASWA 88 thailandensis QV VDPLNDPNSPLAKR HAQYLKSHPQRH+LLALGQKRAEAVRR DSQMEA QNRRADLVYQQ B. LD+ AN− GAVSTQP+++V SIYFDFDSYSVKD+YQPLLQQ QGNTDERSTSEYN AL+L+GV VSLGKEKP LATGHDE +SW 88 graninisAQVNVDPLNDPNSPLAKR H+QYLKSHPQRHVL LALGQKRAEAVRR DSQMEA QNRRADLVYQQ B.LD+ AN−GAVSTQP++NVA SIYFDF DSYSV+D+YQPLLQQ QGNTDERSTSEYN AL+LLGVVSLGKEKP LA GHDEASWA 87 aklahomensis QV VDPLNDPNSPLAKRS HAQYLK HPQRH+LLALGQKRAEAVRR DSQMEA QNRRADLVYQQ B. LD+ AN G−AVSTQP+ +N SIYFDFDSYSV+D+YQ LLQQ QGNTDERSTSEYN AL+LLGV VSLGKEKP LA GHDEASWA 87pseudomallei VAQV VDPLNDPNSPLAKR HAQYLKSHPQRH+L LALGQKRAEAVRR D+QMEAQNRRADLVYQQ B. LD+ AN G−+VS QP+++V SIYFDF DSYSVKD+YQ LLQQ QGNTDERSTSEYNAL+L+GV VSLGKEKP LATGHDE+SWA 86 xenovarans A V VDPLNDPNSPLAKRHAQYLKSHPQRH+L LALGQKRAEAVRR DSQMEA QNRRADLVYQQ B. LD+ AN− G VS QP+ ++SIYFDF DSYSVKD+YQ LLQQ QGNTDERSTSEYN AL+L+GV VSLGKEKP LATGHDE+SWA 86phytafirmans VA VNVDPLNDPNSPLAKR HAQYLKSHPQRHVL LALGQKRAEAVRR DSQMEAQRNRADLVYQQ B. LD+ A − GA QP+ ++VA SIYFDF DSYSVKD+YQPLLQQ QGNTDERSTSEYNAL+LLGV VSLGKEKP LATGHDEASWA 86 phymanon VNVDPLNDPNSPLAKR HAQYLKSHPQRHVLLALGQKRAEAVRR DS+MEA QNRRADLVYQQ R. LDD + + GA− ANVA V+ SIYFDFSDYSVK +YQ +LQ QGNTDERSTSEYN +LA +GV VSLGKEKP ATGHD+ASWA+ 73metallidurans V−DPLNDPNPLAKR H+QVL S+ R+LQ LALGQKRAEAVRR DSQMEA NRRAD+VYR. LDD ANAGA AD− V V+V SIYFDF SDYSVK EYQ +L + QGNTDERSTSEYN +LA +GVVSLGKEKP +TGHDEA+WA+ 73 entropha −DPLNDPNSPLAKR HA+YL S+ R +LLALGQKRAEAVRR +DSQMEA NRRAD+ Y R. LDD−K G +T NV V+V−D SIYFDFDSY+VK EYQ LL Q QGNTDERSTSEYN  AL+ GV VSLGKEKP ATGHDESWAQN 74solanacearan L DPNSPLAKR HA+YL+SH QR VL LALGQKRAEAVRR DSQMEA RR+D+ VYIntranasal Immunization with OMP-NE Induces Anti-OMP Specific Antibodies

The humoral immune responses against the OMP induced by nasal vaccinewith OMP were characterized in vivo in the CD-1 mice. Intranasalvaccination with either 5 μg or 15 μg OMP preparations+NE (OMP-NE)resulted in high serum titers of OMP-specific IgG antibodies of 2.8×10⁵and 5.1×10⁵ at 6 weeks, respectively, following primary vaccination (SeeFIG. 30A). OMPs without adjuvant were immunogenic and resulted in serumanti-OMP IgG titers of 1.9×10⁴ and 3.8×10⁴ six weeks following primaryi.n. immunization. However, treatment groups with NE as an adjuvantresponded significantly higher (13 to 30 fold) than groups withoutadjuvant at all time points following the boost (p<0.05). Further, miceimmunized with OMP in PBS did not demonstrate a significant boost effectfollowing the second vaccination (See FIG. 30A).

To further characterize the OMP-NE vaccine, the OMP-NE composition'sability to elicit specific antibody production in bronchiolar secretionswas evaluated. Mucosal antibody production may be important forprotection against Burkholderia colonization since secretory antibodiesare thought to be critical effectors in protection against mucosalrespiratory pathogens (See, e.g., Nelson et al., 1993, J Med Microbiol39, 39-47). Mucosal immune responses were evaluated in bronchoalveolarlavage (BAL) fluid of animals immunized with 5 μg OMP-NE or 5 μg OMP inPBS. Comparable levels of anti-OMP antibodies in BALs were detected inmice immunized either with OMP-NE or OMP without NE adjuvant (See FIG.30B).

OMP-NE Immunization Yields a Balanced Th1/Th2 Cellular Response

The analysis of the serum IgG subclass was performed to determine the Thelper-type bias of the cellular response. The pattern of IgG subclassdistribution shows that i.n. immunization with OMP-NE produced increasedlevels of Th1-type IgG2b versus Th2-type IgG1 subclass antibodies(p=0.048) (See FIG. 31A). In comparison, immunization with OMP in PBSproduced similar levels of IgG1 and IgG2b antibodies (See FIG. 31A).

OMP-specific cellular responses were characterized in splenocytesobtained 6 weeks following primary vaccination with OMP-NE. The cellswere stimulated with endotoxin-depleted OMP and then evaluated forspecific cytokine production. In splenocytes collected from OMP-NEvaccinated mice, Th1 cytokines IFN-γ and IL-2 production increased 22.1and 18.6-fold respectively over that of splenocytes from non-vaccinatedmice (p=0.01 and 0.003, respectively) (See FIG. 31B). IFN-γ was equallyinduced in splenocytes from mice immunized with OMP in PBS. The Th2cytokines IL-4, IL-5, IL-6, and IL-10 were minimally induced (≦5.22-foldincrease) in both OMP-NE and OMP immunized mice in equivalent amounts.The pattern of splenic cytokine expression and the IgG subclassdistribution results indicated a balanced Th1/Th2 response to OMP-NEvaccination.

Antibody Response is Directed Toward OMP Lipoprotein and LPS

It has been previously documented that LPS is intrinsically associatedwith Burkholderia spp. OMP preparations (See, e.g., Bertot et al., 2007,Infect Immun 75, 2740-2752). To investigate the specificity of immuneresponse and to evaluate the possible immune enhancing role of endotoxinin B. cencocepacia OMP preparations, serum from mice immunized withOMP-NE was analyzed using ELISA with either crude OMP,endotoxin-depleted OMP, or LPS. The highest IgG reactivity was againstthe endotoxin-depleted and crude OMP preparations. The statisticalanalysis indicated a significantly higher response in endotoxin-depletedOMP versus LPS (OD₄₀₅=2.37±0.11 (p=0.001)) and crude OMP versus LPS(OD₄₀₅=1.49±0.14 (p=0.01)). However, the serum also containedsignificant levels of IgG anti-LPS antibodies (OD₄₀₅=0.59±0.05).

To clarify the role of endotoxin in the OMP preparation, a proteolyticdigest of the crude OMP and endotoxin-depleted OMP preparations wereperformed with proteinase K. The immunogenic epitopes were determined bywestern blot analysis comparing serum antibodies generated in micevaccinated with OMP in PBS versus from mice vaccinated with OMP-NE (SeeFIG. 32). The results clearly indicated that antibody response wasdirected mainly toward 62 KDa and 17 KDa proteins contained within theOMP preparation. This was confirmed by the complete absence of theprotein bands in the proteolytically digested OMP preparations (See FIG.32 OMP-NE serum western blot). Further, when the OMP preparations wereprobed with commercial monoclonal anti-LPS antibody, LPS was detected asan intrinsic part of the OMP preparation. However, the proteolyticdigest diminished the LPS detection but not totally. Overall, thepresent invention provides that nasal immunization with OMP-NE enhancesthe production of antibodies specific against OMP proteins (See FIG.32).

Intranasal Vaccination with OMP-NE Results in Cross-ProtectiveNeutralizing Serum Antibody

B. cenocepacia neutralizing antibodies were determined using a colonyreduction assay. The mice intranasally vaccinated with 5 μg or 15 μgOMP-NE produced serum-neutralizing antibodies that inhibited 92.5% or98.3% of B. cenocepacia colonies, respectively, as compared to serumfrom non-vaccinated mice (See FIG. 33). The mice vaccinated with OMPlacking adjuvant also demonstrated a relatively high level ofinhibition, resulting in 33.3% and 46.7% (for 5 μg and 15 μg OMP-PBS,respectively) cfu reduction. The statistical analysis showed that B.cenocepacia growth was significantly inhibited by serum derived fromOMP-NE as compared to serum from those who were immunized with the OMPpreparation alone (See FIG. 33).

To evaluate if the OMP-NE can produce cross-reactive protection fromother bacterial strains, serum from mice vaccinated with 15 μg OMP-NEwas also incubated with B. multivorans. The B. multivorans was selectedbecause it is the most common isolate cultured from CF patients infectedwith Bcc organisms (See, e.g., Baldwin et al., 2008, J Clin Microbiol46, 290-295). The serum inhibited B. multivorans growth by 80.1% and by49.8% derived from mice vaccinated with OMP-NE and OMP without adjuvant,respectively, suggesting significant cross-protective antibodiesfollowing immunization with either OMP-NE or OMP in PBS (See FIG. 33).

Immunization with OMP-NE Protects Against Pulmonary B. cenocepaciaChallenge and Reduces Incidence of Sepsis

The protective effect of intranasal immunization was further tested invivo in a lung infection model. The clearance of B. cenocepacia frompulmonary tissue was evaluated 6 days following intra-trachealinoculation in mice that were intranasally immunized with the OMP-NE orthe OMP without an adjuvant. Vaccination with 15 μg OMP-NE resulted insignificantly higher rates of pulmonary clearance (p=9.2×10⁻³) ascompared to the non-vaccinated mice (See FIG. 34A). At day 6, theaverage pulmonary bacterial load was 22.5±26.2 cfu in mice vaccinatedwith 15 μg OMP-NE, compared to 1.28×10⁶±3.36×10⁶ cfu per lung innon-vaccinated mice, representing a greater than 5-log reduction in thebacterial load.

Splenic colonization following pulmonary inoculation with B. cenocepaciawas evaluated as a means of assessing sepsis (See FIG. 34B). Vaccinationwith 15 μg OMP-NE significantly reduced the incidence of bacteria in thespleens 6 days following the pulmonary challenge of individual mice froman average of 3.54×10³±6.97×10³ cfu per spleen in non-vaccinated mice to2.5±5 cfu per spleen in vaccinates (p=0.0307).

Intranasal Immunization with OMP-NE Attenuates Systemic Illness after B.cenocepacia Infection

B. cenocepacia infection resulted in greater loss of thermoregulation byday 6 in the non-vaccinated mice than in the mice immunized with OMP-NEor OMP without the adjuvant. The mean body temperature 6 days followinginfection with B. cenocepacia was significantly lower in non-vaccinatedmice (35.4° C.±0.7) as compared to mice vaccinated with 15 μg OMP—NE(36.8° C.±0.31, p=0.008), 5 μg OMP-NE (37.1° C.±0.42, p=0.008), or 5 μgOMP without the adjuvant (36.8° C.±0.61, p=0.01).

The total peripheral leukocyte counts determined at the time ofsacrifice in the non-vaccinated mice were significantly higher from thevalues observed for the mice vaccinated with 15 μg OMP-NE (p=0.047), 5μg OMP-NE (p=0.026), or 15 μg OMP in PBS (p=0.032). The ratio ofpolymorphonuclear leukocytes to total peripheral leukocytes were alsosignificantly higher in non-vaccinated mice (76.0%) compared to micevaccinated with 15 μg OMP-NE (47.7%, p=0.02), 5 μg OMP-NE (45.6%,p=0.024), or 15 μg OMP in PBS (48.7%, p=0.04) (See Table 26, below). Incombination with the splenic colonization results described above (SeeFIG. 39B), the present invention provides that immunization with OMP-NEresulted in significantly decreased systemic disease following infectionwith B. cenocepacia.

TABLE 26 Ratio of polymorphonuclear leukocytes to total peripheralleukocytes. Mean Polymorphonuclear Leukocytes Immunization ConcentrationGroup (10⁹/L) % Total Pertipheral Leukocyte Count 5 μg OMP-NE 1.99 ±0.20* 45.6* 5 μg OMP in PBS 3.23 ± 0.29  59.7 15 μg OMP-NE 2.58 ± 0.48*47.7* 15 μg OMP in 2.56 ± 0.55* 48.7* PBS Non-Vaccinated 7.29 ± 1.35 76.0

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions and methods of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe present invention.

1. (canceled)
 2. A method of treating an infection of a wound comprisingadministering a composition comprising a nanoemulsion, or a dilutionthereof, wherein the nanoemulsion comprises:
 1. 30-90%, 60-80%, or60-70% by volume oil;
 2. 3-15% by volume solvent; wherein said solventis selected from the group consisting of ethanol and glycerol; 3.0.05-10.0% by volume non-ionic surfactant and 0.001-5.0% by volumecationic surfactant;
 4. ethylenediaminetetraacetic acid (EDTA); and 5.water to said wound, wherein said composition kills or inhibits growthof bacteria within the wound infection in the absence of increasedinflammation and edema at the site of the wound.
 3. The method of claim1, wherein said wound infection comprises Staphylococcus aureus.
 4. Themethod of claim 3, wherein said Staphylococcus aureus are antibioticresistant.
 5. The method of claim 2, wherein said wound infectioncomprises Pseudomonas aeruginosa.
 6. The method of claim 2, wherein saidcomposition is co-administered with a bioactive agent.
 7. The method ofclaim 2, wherein said nanoemulsion comprises: a) water; b)polyoxyethylenesorbitan monolaurate; c) glycerol or ethanol; d) oil; e)dimethyl benzyl ammonium chloride; and f) ethylenediaminetetraaceticacid (EDTA).
 8. The method of claim 2, wherein said nanoemulsioncomprises: a) water; b) a non-ionic surfactant selected from the groupconsisting of polyoxyethylenesorbitan monolaurate,polyoxyethylenesorbitan monooleate, and a poloxamer; c) glycerol orethanol; d) oil; e) a cationic surfactant selected from the groupconsisting of cetylpyridinium chloride (CPC), benzalkonium chloride, andalkyl dimethyl benzyl ammonium chloride; and f)ethylenediaminetetraacetic acid (EDTA).
 9. The method of claim 2,wherein said nanoemulsion has a mean particle size of 0.2-0.8 microns.10. The method of claim 2, wherein said nanoemulsion has a mean particlesize less than 0.5 microns.
 11. The method of claim 2, wherein saidcomposition is co-administered with an antimicrobial agent.
 12. Themethod of claim 11, wherein said antimicrobial agent is an antibiotic.13. The method of claim 2, wherein said nanoemulsion comprises: a)water; b) 0.05-10.0% polyoxyethylene sorbitan monolaurate; c) 3-15%glycerol; d) oil; e) 0.001-5.0% benzalkonium chloride; and f)ethylenediaminetetraacetic acid (EDTA).